Compositions and methods for inhibiting GYS2 expression

Abstract

This disclosure relates to oligonucleotides, compositions and methods useful for reducing GYS2 expression, particularly in hepatocytes. Disclosed oligonucleotides for the reduction of GYS2 expression may be double-stranded or single-stranded, and may be modified for improved characteristics such as stronger resistance to nucleases and lower immunogenicity. Disclosed oligonucleotides for the reduction of GYS2 expression may also include targeting ligands to target a particular cell or organ, such as the hepatocytes of the liver, and may be used to treat glycogen storage diseases (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) and related conditions.

Description

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/637,574, filed Mar. 2, 2018, and entitled “COMPOSITIONS AND METHODS FOR INHIBITING GYS2 EXPRESSION,” the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present application relates to oligonucleotides and uses thereof, particularly uses relating to the treatment of glycogen storage diseases and associated conditions.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled D0800.70014WO00-SEQ.txt created on Feb. 15, 2019 which is 132 kilobytes in size. The information in electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Glycogen is a complex sugar used by the body to store glucose. When the body requires more glucose to function, it normally breaks down the stored glycogen for use in cellular processes. Several enzymes participate in the processes that are used to store glucose as glycogen (glycogen synthesis) and break down glycogen to glucose (glycogen breakdown). When one or more of these enzymes are inhibited, it can result in a glycogen storage disease in which a dearth of glycogen storage, a buildup of glycogen in affected cells (e.g., liver and/or muscle cells), or the formation of abnormally structured glycogen may be observed. When a disorder of glycogen storage or breakdown occurs, those affected may suffer from a number of symptoms including, but not limited to: hepatomegaly, increased liver toxicity (e.g., higher levels of AST, ALT, and/or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma. A non-limiting set of exemplary glycogen storage diseases may include: GSDI (e.g., GSDIa), GSDIII, GSDIV, GSDVI, and GSDIX.

BRIEF SUMMARY OF THE INVENTION

Aspects of the disclosure relate to oligonucleotides and related methods for treating a glycogen storage disease (e.g., a disease or disorder affecting glycogen breakdown or storage such as GSDIa, GSDIII, GSDIV, GSDVI, or GSDIX) in a subject. In some embodiments, potent RNAi oligonucleotides have been developed for selectively inhibiting GYS2 expression in a subject. In some embodiments, the RNAi oligonucleotides are useful for reducing overall GYS2 activity in hepatocytes, and thereby decreasing or preventing hepatomegaly, liver toxicity (e.g., levels of AST, ALT, and/or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma. In some embodiments, key regions of GYS2 mRNA (referred to as hotspots) have been identified herein that are particularly amenable to targeting using such oligonucleotide-based approaches (See, e.g., Example 1).

One aspect of the present disclosure provides oligonucleotides for reducing expression of GYS2. In some embodiments, the oligonucleotides comprise an antisense strand comprising a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some embodiments, the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some embodiments, the antisense strand comprises, or consists of, a sequence as set forth in any one of SEQ ID NOs: 417-466, 575-580, 586-598, 620-627. In some embodiments, the oligonucleotides further comprise a sense strand that comprises a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the sense strand comprises, or consists of, a sequence as set forth in any one of SEQ ID NOs: 385-416, 569-574, 581-585, 612-619.

One aspect of the present disclosure provides oligonucleotides for reducing expression of GYS2, in which the oligonucleotides comprise an antisense strand of 15 to 30 nucleotides in length. In some embodiments, the antisense strand has a region of complementarity to a target sequence of GYS2 as set forth in any one of SEQ ID NOs: 599-608. In some embodiments, the region of complementarity is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 contiguous nucleotides in length. In some embodiments, the region of complementarity is fully complementary to the target sequence of GYS2. In some embodiments, the region of complementarity to GYS2 is at least 19 contiguous nucleotides in length.

In some embodiments, the antisense strand is 19 to 27 nucleotides in length. In some embodiments, the antisense strand is 21 to 27 nucleotides in length. In some embodiments, the oligonucleotide further comprises a sense strand of 15 to 40 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand. In some embodiments, the sense strand is 19 to 40 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 25 nucleotides in length. In some embodiments, the duplex region is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides in length. In some embodiments, the antisense strand and sense strand form a duplex region of 25 nucleotides in length.

In some embodiments, an oligonucleotide comprises an antisense strand and a sense strand that are each in a range of 21 to 23 nucleotides in length. In some embodiments, an oligonucleotide comprises a duplex structure in a range of 19 to 21 nucleotides in length. In some embodiments, an oligonucleotide comprises a 3′-overhang sequence of one or more nucleotides in length, in which the 3′-overhang sequence is present on the antisense strand, the sense strand, or the antisense strand and sense strand. In some embodiments, an oligonucleotide further comprises a 3′-overhang sequence on the antisense strand of two nucleotides in length. In some embodiments, an oligonucleotide comprises a 3′-overhang sequence of two nucleotides in length, in which the 3′-overhang sequence is present on the antisense strand, and in which the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and antisense strand form a duplex of 21 nucleotides in length.

In some embodiments, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some embodiments, the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627.

In some embodiments, the sense strand comprises at its 3′-end a stem-loop set forth as: S1-L-S2, in which S1 is complementary to S2, and in which L forms a loop between S1 and S2 of 3 to 5 nucleotides in length.

Another aspect of the present disclosure provides an oligonucleotide for reducing expression of GYS2, the oligonucleotide comprising an antisense strand and a sense strand, in which the antisense strand is 21 to 27 nucleotides in length and has a region of complementarity to GYS2, in which the sense strand comprises at its 3′-end a stem-loop set forth as: S1-L-S2, in which S1 is complementary to S2, and in which L forms a loop between S1 and S2 of 3 to 5 nucleotides in length, and in which the antisense strand and the sense strand form a duplex structure of at least 19 nucleotides in length but are not covalently linked (see, e.g., FIG. 3). In some embodiments, the region of complementarity is fully complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides of GYS2 mRNA. In some embodiments, L is a tetraloop. In some embodiments, L is 4 nucleotides in length. In some embodiments, L comprises a sequence set forth as GAAA.

In some embodiments, an oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a 2′-modification. In some embodiments, the 2′-modification is a modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid. In some embodiments, all of the nucleotides of an oligonucleotide are modified.

In some embodiments, an oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog. In some embodiments, the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

In some embodiments, at least one nucleotide of an oligonucleotide is conjugated to one or more targeting ligands. In some embodiments, each targeting ligand comprises a carbohydrate, amino sugar, cholesterol, polypeptide, or lipid. In some embodiments, each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety. In some embodiments, up to 4 nucleotides of L of the stem-loop are each conjugated to a monovalent GalNAc moiety. In some embodiments, the targeting ligand comprises an aptamer.

Another aspect of the present disclosure provides a composition comprising an oligonucleotide of the present disclosure and an excipient. Another aspect of the present disclosure provides a method comprising administering a composition of the present disclosure to a subject. In some embodiments, the method results in a decreased level or prevention of hepatomegaly, liver nodule formation, liver toxicity (e.g., levels of AST, ALT, and/or ALP), liver fibrosis, hepatocellular proliferation, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma. Another aspect of the present disclosure provides a method for treating a glycogen storage disease or one or more symptoms of a glycogen storage disease. A non-limiting set of exemplary glycogen storage diseases may include: GSDI (e.g., GSDIa), GSDIII, GSDIV, GSDVI, and GSDIX.

Another aspect of the present disclosure provides an oligonucleotide for reducing expression of GYS2, the oligonucleotide comprising a sense strand of 15 to 40 nucleotides in length and an antisense strand of 15 to 30 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand, in which the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619, and results in a the antisense strand comprises a complementary sequence selected from SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627.

In some embodiments, the oligonucleotide comprises a pair of sense and antisense strands selected from a row of the table set forth in Table 4.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to provide non-limiting examples of certain aspects of the compositions and methods disclosed herein.

FIGS. 1A and 1B are graphs showing the percentage of GYS2 mRNA remaining after a screen of 264 GYS2 conjugates in HEK-293 cells. The nucleotide position in NM_021957.3 that corresponds to the 3′ end of the sense strand of each siRNA is indicated on the x axis.

FIGS. 2A and 2B is a set of graphs showing the percentage of mRNA remaining after GYS2 oligonucleotide screening of 71 GYS2 oligonucleotides at two or three different concentrations (0.1 nM and 1.0 nM or 0.03 nM, 0.1 nM, and 1.0 nM) in HEK-293 cells.

FIG. 3 is a schematic showing a non-limiting example of a double-stranded oligonucleotide with a nicked tetraloop structure that has been conjugated to four GalNAc moieties (diamond shapes).

FIG. 4 is a graph showing the results of screening in HEK-293 cells using GYS2 oligonucleotides of different base sequences in one or two different modification patterns. The X-axis lists the 3′ end of the sense strand targeted by the oligonucleotide evaluated. A negative control sequence (NCI), untransfected cells, and mock transfected cells are shown at left as controls.

FIG. 5 is a graph showing the results of screening in monkey hepatocyte cells using GYS2 oligonucleotides of different base sequences in the nicked tetraloop structure. The same modification pattern was used, and the oligonucleotides were tested at three different concentrations (0.1 μM, 0.3 μM, and 1.0 μM). Untransfected cells are shown as a control at left.

FIGS. 6A and 6B are a series of graphs showing the IC₅₀ results for GYS2 oligonucleotides selected from dose response curve screening in HEK-293 cells.

FIG. 7 is a graph showing an in vivo activity evaluation of GalNAc-conjugated GYS2 oligonucleotides in a nicked tetraloop structure. Eight different oligonucleotide sequences were tested. Oligonucleotides were subcutaneously administered to mice expressed human GYS2, at 0.5 mg/kg. The data show the amount of GYS2 mRNA remaining at day 4 following administration normalized to PBS control.

DETAILED DESCRIPTION OF THE INVENTION

According to some aspects, the disclosure provides oligonucleotides targeting GYS2 mRNA that are effective for reducing GYS2 expression in cells, particularly liver cells (e.g., hepatocytes) for the treatment of a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) or one or more symptoms of a glycogen storage disease. Accordingly, in related aspects, the disclosure provided methods of treating a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) or one or more symptoms of a glycogen storage disease that involve selectively reducing GYS2 gene expression in liver. In certain embodiments, GYS2 targeting oligonucleotides provided herein are designed for delivery to selected cells of target tissues (e.g., liver hepatocytes) to treat a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) or one or more symptoms of a glycogen storage disease in a subject.

Further aspects of the disclosure, including a description of defined terms, are provided below.

I. Definitions

Administering: As used herein, the terms “administering” or “administration” means to provide a substance (e.g., an oligonucleotide) to a subject in a manner that is pharmacologically useful (e.g., to treat a condition in the subject).

Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Asialoglycoprotein receptor (ASGPR): As used herein, the term “Asialoglycoprotein receptor” or “ASGPR” refers to a bipartite C-type lectin formed by a major 48 kDa (ASGPR-1) and minor 40 kDa subunit (ASGPR-2). ASGPR is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins).

Complementary: As used herein, the term “complementary” refers to a structural relationship between nucleotides (e.g., two nucleotide on opposing nucleic acids or on opposing regions of a single nucleic acid strand) that permits the nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. In some embodiments, complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. In some embodiments, two nucleic acids may have nucleotide sequences that are complementary to each other so as to form regions of complementarity, as described herein.

Deoxyribonucleotide: As used herein, the term “deoxyribonucleotide” refers to a nucleotide having a hydrogen at the 2′ position of its pentose sugar as compared with a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions of atoms other than at the 2′ position, including modifications or substitutions in or of the sugar, phosphate group or base.

Double-stranded oligonucleotide: As used herein, the term “double-stranded oligonucleotide” refers to an oligonucleotide that is substantially in a duplex form. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides of covalently separate nucleic acid strands. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides of nucleic acid strands that are covalently linked. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed from a single nucleic acid strand that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that base pair together. In some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are fully duplexed with one another. However, in some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are partially duplexed, e.g., having overhangs at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary, and thus, may have one or more mismatches, which may include internal mismatches or end mismatches.

Duplex: As used herein, the term “duplex,” in reference to nucleic acids (e.g., oligonucleotides), refers to a structure formed through complementary base-pairing of two antiparallel sequences of nucleotides.

Excipient: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect.

Glycogen Storage Disease: As used herein, the term “glycogen storage disease,” “GSD,” or “glycogen storage diseases” refers to metabolic disorders caused by enzyme deficiencies affecting glycogen synthesis, glycogen breakdown, and/or glucose breakdown (glycolysis). Various types of glycogen storage diseases have been characterized, including GSD 0, GSD I (also known as GSD 1 or von Gierke's disease; e.g., GSDIa), GSD II (also known as Pompe disease or acid maltase deficiency disease), GSD III (also known as GSD 3, Cori's disease, or Forbes' disease), GSD IV (GSD 4 or Andersen disease), GSD V (also known as McArdle disease), GSD VI (also known as GSD 6 or Hers' disease), GSD VII (also known as GSD 7 or Tarui's disease), GSD VIII, and GSD IX (also known as GSD 9). In some embodiments, individuals having a glycogen storage disease exhibit one or more of a number of symptoms including, but not limited to: hepatomegaly, increased liver toxicity (e.g., higher levels of AST, ALT, and/or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma.

GYS2: as used herein, the term “GYS2” or “glycogen synthase 2” refers to the liver glycogen synthase gene. This gene encodes a protein, liver glycogen synthase, that catalyzes a rate-limiting stem in the synthesis of glycogen (i.e., the transfer of a glucose molecule from UDP-glucose to a terminal branch of the glycogen molecule). GYS2 is expressed in liver cells, e.g., hepatocytes. Homologs of GYS2 are conserved across a range of species, including human, mouse, rat, non-human primate species, and others (see, e.g., NCBI HomoloGene: 56580.) In humans, GYS2 encodes multiple transcripts, namely as set forth in GenBank accession numbers NM_021957.3 (SEQ ID NO: 609), XM_006719063.3, and XM_017019245.1, each encoding a different isoform, GenBank accession numbers NP_068776.2, XP_006719126.1 (isoform X1) and XP 016874734.1 (isoform X2), respectively. An example monkey (Rhesus macaque) transcript sequence is set forth in GenBank accession number XM_001098578.2 (SEQ ID NO: 610). An example mouse transcript is set forth in GenBank accession number NM_145572.2 (SEQ ID NO: 611).

Hepatocyte: As used herein, the term “hepatocyte” or “hepatocytes” refers to cells of the parenchymal tissues of the liver. These cells make up approximately 70-85% of the liver's mass and manufacture serum albumin, fibrinogen, and the prothrombin group of clotting factors (except for Factors 3 and 4). Markers for hepatocyte lineage cells may include, but are not limited to: transthyretin (Ttr), glutamine synthetase (Glul), hepatocyte nuclear factor 1a (Hnf1a), and hepatocyte nuclear factor 4a (Hnf4a). Markers for mature hepatocytes may include, but are not limited to: cytochrome P450 (Cyp3a11), fumarylacetoacetate hydrolase (Fah), glucose 6-phosphate (G6p), albumin (Alb), and OC2-2F8. See, e.g., Huch et al., (2013), Nature, 494(7436): 247-250, the contents of which relating to hepatocyte markers is incorporated herein by reference.

Loop: As used herein, the term “loop” refers to an unpaired region of a nucleic acid (e.g., oligonucleotide) that is flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to one another, such that under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cells), the two antiparallel regions, which flank the unpaired region, hybridize to form a duplex (referred to as a “stem”).

Modified Internucleotide Linkage: As used herein, the term “modified internucleotide linkage” refers to an internucleotide linkage having one or more chemical modifications compared with a reference internucleotide linkage comprising a phosphodiester bond. In some embodiments, a modified nucleotide is a non-naturally occurring linkage. Typically, a modified internucleotide linkage confers one or more desirable properties to a nucleic acid in which the modified internucleotide linkage is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.

Modified Nucleotide: As used herein, the term “modified nucleotide” refers to a nucleotide having one or more chemical modifications compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide and thymidine deoxyribonucleotide. In some embodiments, a modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, a modified nucleotide has one or more chemical modifications in its sugar, nucleobase and/or phosphate group. In some embodiments, a modified nucleotide has one or more chemical moieties conjugated to a corresponding reference nucleotide. Typically, a modified nucleotide confers one or more desirable properties to a nucleic acid in which the modified nucleotide is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc. In certain embodiments, a modified nucleotide comprises a 2′-O-methyl or a 2′-F substitution at the 2′ position of the ribose ring.

Nicked Tetraloop Structure: A “nicked tetraloop structure” is a structure of a RNAi oligonucleotide characterized by the presence of separate sense (passenger) and antisense (guide) strands, in which the sense strand has a region of complementarity to the antisense strand such that the two strands form a duplex, and in which at least one of the strands, generally the sense strand, extends from the duplex in which the extension contains a tetraloop and two self-complementary sequences forming a stem region adjacent to the tetraloop, in which the tetraloop is configured to stabilize the adjacent stem region formed by the self-complementary sequences of the at least one strand.

Oligonucleotide: As used herein, the term “oligonucleotide” refers to a short nucleic acid, e.g., of less than 100 nucleotides in length. An oligonucleotide can comprise ribonucleotides, deoxyribonucleotides, and/or modified nucleotides including, for example, modified ribonucleotides. An oligonucleotide may be single-stranded or double-stranded. An oligonucleotide may or may not have duplex regions. As a set of non-limiting examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA, or single-stranded siRNA. In some embodiments, a double-stranded oligonucleotide is an RNAi oligonucleotide.

Overhang: As used herein, the term “overhang” refers to terminal non-base-pairing nucleotide(s) resulting from one strand or region extending beyond the terminus of a complementary strand with which the one strand or region forms a duplex. In some embodiments, an overhang comprises one or more unpaired nucleotides extending from a duplex region at the 5′ terminus or 3′ terminus of a double-stranded oligonucleotide. In certain embodiments, the overhang is a 3′ or 5′ overhang on the antisense strand or sense strand of a double-stranded oligonucleotide.

Phosphate analog: As used herein, the term “phosphate analog” refers to a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5′ terminal nucleotide of an oligonucleotide in place of a 5′-phosphate, which is often susceptible to enzymatic removal. In some embodiments, a 5′ phosphate analog contains a phosphatase-resistant linkage. Examples of phosphate analogs include 5′ phosphonates, such as 5′ methylenephosphonate (5′-MP) and 5′-(E)-vinylphosphonate (5′-VP). In some embodiments, an oligonucleotide has a phosphate analog at a 4′-carbon position of the sugar (referred to as a “4′-phosphate analog”) at a 5′-terminal nucleotide. An example of a 4′-phosphate analog is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof. See, for example, International Patent Application PCT/US2017/049909, filed on Sep. 1, 2017, U.S. Provisional Application Nos. 62/383,207, filed on Sep. 2, 2016, and 62/393,401, filed on Sep. 12, 2016, the contents of each of which relating to phosphate analogs are incorporated herein by reference. Other modifications have been developed for the 5′ end of oligonucleotides (see, e.g., WO 2011/133871; U.S. Pat. No. 8,927,513; and Prakash et al. (2015), Nucleic Acids Res., 43(6):2993-3011, the contents of each of which relating to phosphate analogs are incorporated herein by reference).

Reduced expression: As used herein, the term “reduced expression” of a gene refers to a decrease in the amount of RNA transcript or protein encoded by the gene and/or a decrease in the amount of activity of the gene in a cell or subject, as compared to an appropriate reference cell or subject. For example, the act of treating a cell with a double-stranded oligonucleotide (e.g., one having an antisense strand that is complementary to GYS2 mRNA sequence) may result in a decrease in the amount of RNA transcript, protein and/or enzymatic activity (e.g., encoded by the GYS2 gene) compared to a cell that is not treated with the double-stranded oligonucleotide. Similarly, “reducing expression” as used herein refers to an act that results in reduced expression of a gene (e.g., GYS2).

Region of Complementarity: As used herein, the term “region of complementarity” refers to a sequence of nucleotides of a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel sequence of nucleotides (e.g., a target nucleotide sequence within an mRNA) to permit hybridization between the two sequences of nucleotides under appropriate hybridization conditions, e.g., in a phosphate buffer, in a cell, etc. A region of complementarity may be fully complementary to a nucleotide sequence (e.g., a target nucleotide sequence present within an mRNA or portion thereof). For example, a region of complementary that is fully complementary to a nucleotide sequence present in an mRNA has a contiguous sequence of nucleotides that is complementary, without any mismatches or gaps, to a corresponding sequence in the mRNA. Alternatively, a region of complementarity may be partially complementary to a nucleotide sequence (e.g., a nucleotide sequence present in an mRNA or portion thereof). For example, a region of complementary that is partially complementary to a nucleotide sequence present in an mRNA has a contiguous sequence of nucleotides that is complementary to a corresponding sequence in the mRNA but that contains one or more mismatches or gaps (e.g., 1, 2, 3, or more mismatches or gaps) compared with the corresponding sequence in the mRNA, provided that the region of complementarity remains capable of hybridizing with the mRNA under appropriate hybridization conditions.

Ribonucleotide: As used herein, the term “ribonucleotide” refers to a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2′ position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than at the 2′ position, including modifications or substitutions in or of the ribose, phosphate group or base.

RNAi Oligonucleotide: As used herein, the term “RNAi oligonucleotide” refers to either (a) a double stranded oligonucleotide having a sense strand (passenger) and antisense strand (guide), in which the antisense strand or part of the antisense strand is used by the Argonaute 2 (Ago2) endonuclease in the cleavage of a target mRNA or (b) a single stranded oligonucleotide having a single antisense strand, where that antisense strand (or part of that antisense strand) is used by the Ago2 endonuclease in the cleavage of a target mRNA.

Strand: As used herein, the term “strand” refers to a single contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages, phosphorothioate linkages). In some embodiments, a strand has two free ends, e.g., a 5′-end and a 3′-end.

Subject: As used herein, the term “subject” means any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human or non-human primate. The terms “individual” or “patient” may be used interchangeably with “subject.”

Synthetic: As used herein, the term “synthetic” refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid state nucleic acid synthesizer)) or that is otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

Targeting ligand: As used herein, the term “targeting ligand” refers to a molecule (e.g., a carbohydrate, amino sugar, cholesterol, polypeptide or lipid) that selectively binds to a cognate molecule (e.g., a receptor) of a tissue or cell of interest and that is conjugatable to another substance for purposes of targeting the other substance to the tissue or cell of interest. For example, in some embodiments, a targeting ligand may be conjugated to an oligonucleotide for purposes of targeting the oligonucleotide to a specific tissue or cell of interest. In some embodiments, a targeting ligand selectively binds to a cell surface receptor. Accordingly, in some embodiments, a targeting ligand when conjugated to an oligonucleotide facilitates delivery of the oligonucleotide into a particular cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of the complex comprising the oligonucleotide, targeting ligand and receptor. In some embodiments, a targeting ligand is conjugated to an oligonucleotide via a linker that is cleaved following or during cellular internalization such that the oligonucleotide is released from the targeting ligand in the cell.

Tetraloop: As used herein, the term “tetraloop” refers to a loop that increases stability of an adjacent duplex formed by hybridization of flanking sequences of nucleotides. The increase in stability is detectable as an increase in melting temperature (T_m) of an adjacent stem duplex that is higher than the T_m of the adjacent stem duplex expected, on average, from a set of loops of comparable length consisting of randomly selected sequences of nucleotides. For example, a tetraloop can confer a melting temperature of at least 50° C., at least 55° C., at least 56° C., at least 58° C., at least 60° C., at least 65° C., or at least 75° C. in 10 mM NaHPO₄ to a hairpin comprising a duplex of at least 2 base pairs in length. In some embodiments, a tetraloop may stabilize a base pair in an adjacent stem duplex by stacking interactions. In addition, interactions among the nucleotides in a tetraloop include, but are not limited to: non-Watson-Crick base-pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug. 16; 346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12; 253(5016):191-4). In some embodiments, a tetraloop comprises or consists of 3 to 6 nucleotides, and is typically 4 to 5 nucleotides. In certain embodiments, a tetraloop comprises or consists of three, four, five, or six nucleotides, which may or may not be modified (e.g., which may or may not be conjugated to a targeting moiety). In one embodiment, a tetraloop consists of four nucleotides. Any nucleotide may be used in the tetraloop and standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish-Bowden (1985) Nucl. Acids Res. 13: 3021-3030. For example, the letter “N” may be used to mean that any base may be in that position, the letter “R” may be used to show that A (adenine) or G (guanine) may be in that position, and “B” may be used to show that C (cytosine), G (guanine), or T (thymine) may be in that position. Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al., Proc Natl Acad Sci USA. 1990 November; 87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 Nov. 11; 19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA)), the d(GNRA) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). See, for example: Nakano et al. Biochemistry, 41 (48), 14281-14292, 2002. SHINJI et al. Nippon Kagakkai Koen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000), which are incorporated by reference herein for their relevant disclosures. In some embodiments, the tetraloop is contained within a nicked tetraloop structure.

Treat: As used herein, the term “treat” refers to the act of providing care to a subject in need thereof, e.g., through the administration a therapeutic agent (e.g., an oligonucleotide) to the subject, for purposes of improving the health and/or well-being of the subject with respect to an existing condition (e.g., a disease, disorder) or to prevent or decrease the likelihood of the occurrence of a condition. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom or contributing factor of a condition (e.g., disease, disorder) experienced by a subject.

II. Oligonucleotide-Based Inhibitors

i. GYS2 Targeting Oligonucleotides

Potent oligonucleotides have been identified herein through examination of the GYS2 mRNA, including mRNAs of different species (human and Rhesus macaque, (see, e.g., Example 1)) and in vitro and in vivo testing. Such oligonucleotides can be used to achieve therapeutic benefit for subjects with a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) or one or more symptoms of a glycogen storage disease by reducing GYS2 activity, and consequently, by decreasing or preventing hepatomegaly, liver toxicity (demonstrated, e.g., levels of AST, ALT, and/or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma. For example, potent RNAi oligonucleotides are provided herein that have a sense strand comprising, or consisting of, a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619, and an antisense strand comprising, or consisting of, a complementary sequence selected from SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627, as is also arranged the table provided in Table 4 (e.g., a sense strand comprising a sequence as set forth in SEQ ID NO: 1 and an antisense strand comprising a sequence as set forth in SEQ ID NO: 193).

The sequences can be put into multiple different oligonucleotide structures (or formats) as described herein.

In some embodiments, it has been discovered that certain regions of GYS2 mRNA are hotspots for targeting because they are more amenable than other regions to oligonucleotide-based inhibition. In some embodiments, a hotspot region of GYS2 consists of a sequence as forth in any one of SEQ ID NOs: 599-608. These regions of GYS2 mRNA may be targeted using oligonucleotides as discussed herein for purposes of inhibiting GYS2 mRNA expression.

Accordingly, in some embodiments, oligonucleotides provided herein are designed so as to have regions of complementarity to GYS2 mRNA (e.g., within a hotspot of GYS2 mRNA) for purposes of targeting the mRNA in cells and inhibiting its expression. The region of complementarity is generally of a suitable length and base content to enable annealing of the oligonucleotide (or a strand thereof) to GYS2 mRNA for purposes of inhibiting its expression.

In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is at least partially complementary to a sequence as set forth in SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619, which include sequences mapping to within hotspot regions of GYS2 mRNA. In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is fully complementary to a sequence as set forth in SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, a region of complementarity of an oligonucleotide that is complementary to contiguous nucleotides of a sequence as set forth in SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619 spans the entire length of an antisense strand. In some embodiments, a region of complementarity of an oligonucleotide that is complementary to contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619 spans a portion of the entire length of an antisense strand (e.g., all but two nucleotides at the 3′ end of the antisense strand). In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is at least partially (e.g., fully) complementary to a contiguous stretch of nucleotides spanning nucleotides 1-19 of a sequence as set forth in SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619.

In some embodiments, the region of complementarity is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length. In some embodiments, an oligonucleotide provided herein has a region of complementarity to GYS2 mRNA that is in the range of 12 to 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides in length. In some embodiments, an oligonucleotide provided herein has a region of complementarity to GYS2 mRNA that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, a region of complementarity to GYS2 mRNA may have one or more mismatches compared with a corresponding sequence of GYS2 mRNA. A region of complementarity on an oligonucleotide may have up to 1, up to 2, up to 3, up to 4 etc. mismatches provided that it maintains the ability to form complementary base pairs with GYS2 mRNA under appropriate hybridization conditions. Alternatively, a region of complementarity on an oligonucleotide may have no more than 1, no more than 2, no more than 3, or no more than 4 mismatches provided that it maintains the ability to form complementary base pairs with GYS2 mRNA under appropriate hybridization conditions. In some embodiments, if there are more than one mismatches in a region of complementarity, they may be positioned consecutively (e.g., 2, 3, 4, or more in a row), or interspersed throughout the region of complementarity provided that the oligonucleotide maintains the ability to form complementary base pairs with GYS2 mRNA under appropriate hybridization conditions.

Still, in some embodiments, double-stranded oligonucleotides provided herein comprise, or consist of, a sense strand having a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619 and an antisense strand having a complementary sequence selected from SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627, as is arranged in the table provided in Table 4 (e.g., a sense strand comprising a sequence as set forth in SEQ ID NO: 1 and an antisense strand comprising a sequence as set forth in SEQ ID NO: 193).

ii. Oligonucleotide Structures

There are a variety of structures of oligonucleotides that are useful for targeting GYS2 mRNA in the methods of the present disclosure, including RNAi, miRNA, etc. Any of the structures described herein or elsewhere may be used as a framework to incorporate or target a sequence described herein (e.g., a hotpot sequence of GYS2 such as those illustrated in SEQ ID NOs: 599-608, or a sense or antisense strand that comprises or consists of a sequence as set forth SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619 or as set forth SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627, respectively). Double-stranded oligonucleotides for targeting GYS2 expression (e.g., via the RNAi pathway) generally have a sense strand and an antisense strand that form a duplex with one another. In some embodiments, the sense and antisense strands are not covalently linked. However, in some embodiments, the sense and antisense strands are covalently linked.

In some embodiments, sequences described herein can be incorporated into, or targeted using, oligonucleotides that comprise sense and antisense strands that are both in the range of 17 to 40 nucleotides in length. In some embodiments, oligonucleotides incorporating such sequences are provided that have a tetraloop structure within a 3′ extension of their sense strand, and two terminal overhang nucleotides at the 3′ end of its antisense strand. In some embodiments, the two terminal overhang nucleotides are GG. Typically, one or both of the two terminal GG nucleotides of the antisense strand is or are not complementary to the target.

In some embodiments, oligonucleotides incorporating such sequences are provided that have sense and antisense strands that are both in the range of 21 to 23 nucleotides in length. In some embodiments, a 3′ overhang is provided on the sense, antisense, or both sense and antisense strands that is 1 or 2 nucleotides in length. In some embodiments, an oligonucleotide has a guide strand of 23 nucleotides and a passenger strand of 21 nucleotides, in which the 3′-end of passenger strand and 5′-end of guide strand form a blunt end and where the guide strand has a two nucleotide 3′ overhang.

In some embodiments, double-stranded oligonucleotides for reducing GYS2 expression engage RNA interference (RNAi). For example, RNAi oligonucleotides have been developed with each strand having sizes of 19-25 nucleotides with at least one 3′ overhang of 1 to 5 nucleotides (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides have also been developed that are processed by Dicer to generate active RNAi products (see, e.g., U.S. Pat. No. 8,883,996). Further work produced extended double-stranded oligonucleotides where at least one end of at least one strand is extended beyond a duplex targeting region, including structures where one of the strands includes a thermodynamically-stabilizing tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, as well as WO2010033225, which are incorporated by reference herein for their disclosure of these oligonucleotides). Such structures may include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions.

In some embodiments, oligonucleotides may be in the range of 21 to 23 nucleotides in length. In some embodiments, oligonucleotides may have an overhang (e.g., of 1, 2, or 3 nucleotides in length) in the 3′ end of the sense and/or antisense strands. In some embodiments, oligonucleotides (e.g., siRNAs) may comprise a 21 nucleotide guide strand that is antisense to a target RNA and a complementary passenger strand, in which both strands anneal to form a 19-bp duplex and 2 nucleotide overhangs at either or both 3′ ends. See, for example, U.S. Pat. Nos. 9,012,138, 9,012,621, and 9,193,753, the contents of each of which are incorporated herein for their relevant disclosures.

In some embodiments, an oligonucleotide of the invention has a 36 nucleotide sense strand that comprises an region extending beyond the antisense-sense duplex, where the extension region has a stem-tetraloop structure where the stem is a six base pair duplex and where the tetraloop has four nucleotides. In some embodiments, the stem-tetraloop is set forth as: S₁-L-S₂, in which S₁ is complementary to S₂ so as to form a duplex, and in which L forms a tetraloop between S₁ and S₂.

In certain of those embodiments, three or four of the tetraloop nucleotides are each conjugated to a monovalent GalNac ligand.

In some embodiments, an oligonucleotide of the invention comprises a 25 nucleotide sense strand and a 27 nucleotide antisense strand that when acted upon by a dicer enzyme results in an antisense strand that is incorporated into the mature RISC.

Other oligonucleotides designs for use with the compositions and methods disclosed herein include: 16-mer siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., having 19 bp or shorter stems; see, e.g., Moore et al. Methods Mol. Biol. 2010; 629:141-158), blunt siRNAs (e.g., of 19 bps in length; see: e.g., Kraynack and Baker, RNA Vol. 12, p 163-176 (2006)), asymmetrical siRNAs (aiRNA; see, e.g., Sun et al., Nat. Biotechnol. 26, 1379-1382 (2008)), asymmetric shorter-duplex siRNA (see, e.g., Chang et al., Mol Ther. 2009 April; 17(4): 725-32), fork siRNAs (see, e.g., Hohjoh, FEBS Letters, Vol 557, issues 1-3; January 2004, p 193-198), single-stranded siRNAs (Elsner; Nature Biotechnology 30, 1063 (2012)), dumbbell-shaped circular siRNAs (see, e.g., Abe et al. J Am Chem Soc 129: 15108-15109 (2007)), and small internally segmented interfering RNA (sisiRNA; see, e.g., Bramsen et al., Nucleic Acids Res. 2007 September; 35(17): 5886-5897). Each of the foregoing references is incorporated by reference in its entirety for the related disclosures therein. Further non-limiting examples of an oligonucleotide structures that may be used in some embodiments to reduce or inhibit the expression of GYS2 are microRNA (miRNA), short hairpin RNA (shRNA), and short siRNA (see, e.g., Hamilton et al., Embo J., 2002, 21(17): 4671-4679; see also U.S. Application No. 20090099115).

a. Antisense Strands

In some embodiments, an oligonucleotide disclosed herein for targeting GYS2 comprises an antisense strand comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some embodiments, an oligonucleotide comprises an antisense strand comprising or consisting of at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627.

In some embodiments, a double-stranded oligonucleotide may have an antisense strand of up to 40 nucleotides in length (e.g., up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide may have an antisense strand of at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, an oligonucleotide may have an antisense strand in a range of 12 to 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, an oligonucleotide may have an antisense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

In some embodiments, an antisense strand of an oligonucleotide may be referred to as a “guide strand.” For example, if an antisense strand can engage with RNA-induced silencing complex (RISC) and bind to an Argonaut protein, or engage with or bind to one or more similar factors, and direct silencing of a target gene, it may be referred to as a guide strand. In some embodiments, a sense strand complementary to a guide strand may be referred to as a “passenger strand.”

b. Sense Strands

In some embodiments, an oligonucleotide disclosed herein for targeting GYS2 comprises or consists of a sense strand sequence as set forth in in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, an oligonucleotide has a sense strand that comprises or consists of at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619.

In some embodiments, an oligonucleotide may have a sense strand (or passenger strand) of up to 40 nucleotides in length (e.g., up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand of at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand in a range of 12 to 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, an oligonucleotide may have a sense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

In some embodiments, a sense strand comprises a stem-loop structure at its 3′-end. In some embodiments, a sense strand comprises a stem-loop structure at its 5′-end. In some embodiments, a stem is a duplex of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 base pairs in length. In some embodiments, a stem-loop provides the molecule better protection against degradation (e.g., enzymatic degradation) and facilitates targeting characteristics for delivery to a target cell. For example, in some embodiments, a loop provides added nucleotides on which modification can be made without substantially affecting the gene expression inhibition activity of an oligonucleotide. In certain embodiments, an oligonucleotide is provided herein in which the sense strand comprises (e.g., at its 3′-end) a stem-loop set forth as: S₁-L-S₂, in which S₁ is complementary to S₂, and in which L forms a loop between S₁ and S₂ of up to 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length).

In some embodiments, a loop (L) of a stem-loop is a tetraloop (e.g., within a nicked tetraloop structure). A tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a tetraloop has 4 to 5 nucleotides.

c. Duplex Length

In some embodiments, a duplex formed between a sense and antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, a duplex formed between a sense and antisense strand is in the range of 12-30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length). In some embodiments, a duplex formed between a sense and antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments a duplex formed between a sense and antisense strand does not span the entire length of the sense strand and/or antisense strand. In some embodiments, a duplex between a sense and antisense strand spans the entire length of either the sense or antisense strands. In certain embodiments, a duplex between a sense and antisense strand spans the entire length of both the sense strand and the antisense strand.

d. Oligonucleotide Ends

In some embodiments, an oligonucleotide provided herein comprises sense and antisense strands, such that there is a 3′-overhang on either the sense strand or the antisense strand, or both the sense and antisense strand. In some embodiments, oligonucleotides provided herein have one 5′end that is thermodynamically less stable compared to the other 5′ end. In some embodiments, an asymmetric oligonucleotide is provided that includes a blunt end at the 3′ end of a sense strand and an overhang at the 3′ end of an antisense strand. In some embodiments, a 3′ overhang on an antisense strand is 1-8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length).

Typically, an oligonucleotide for RNAi has a two nucleotide overhang on the 3′ end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, an overhang is a 3′ overhang comprising a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides. However, in some embodiments, the overhang is a 5′ overhang comprising a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides.

In some embodiments, one or more (e.g., 2, 3, 4) terminal nucleotides of the 3′ end or 5′ end of a sense and/or antisense strand are modified. For example, in some embodiments, one or two terminal nucleotides of the 3′ end of an antisense strand are modified. In some embodiments, the last nucleotide at the 3′ end of an antisense strand is modified, e.g., comprises 2′-modification, e.g., a 2′-O-methoxyethyl. In some embodiments, the last one or two terminal nucleotides at the 3′ end of an antisense strand are complementary to the target. In some embodiments, the last one or two nucleotides at the 3′ end of the antisense strand are not complementary to the target. In some embodiments, the 5′ end and/or the 3′ end of a sense or antisense strand has an inverted cap nucleotide.

e. Mismatches

In some embodiments, there is one or more (e.g., 1, 2, 3, or 4) mismatches between a sense and antisense strand. If there is more than one mismatch between a sense and antisense strand, they may be positioned consecutively (e.g., 2, 3 or more in a row), or interspersed throughout the region of complementarity. In some embodiments, the 3′-terminus of the sense strand contains one or more mismatches. In one embodiment, two mismatches are incorporated at the 3′ terminus of the sense strand. In some embodiments, base mismatches or destabilization of segments at the 3′-end of the sense strand of the oligonucleotide improved the potency of synthetic duplexes in RNAi, possibly through facilitating processing by Dicer.

iii. Single-Stranded Oligonucleotides

In some embodiments, an oligonucleotide for reducing GYS2 expression as described herein is single-stranded. Such structures may include, but are not limited to single-stranded RNAi oligonucleotides. Recent efforts have demonstrated the activity of single-stranded RNAi oligonucleotides (see, e.g., Matsui et al. (May 2016), Molecular Therapy, Vol. 24(5), 946-955). However, in some embodiments, oligonucleotides provided herein are antisense oligonucleotides (ASOs). An antisense oligonucleotide is a single-stranded oligonucleotide that has a nucleobase sequence which, when written in the 5′ to 3′ direction, comprises the reverse complement of a targeted segment of a particular nucleic acid and is suitably modified (e.g., as a gapmer) so as to induce RNaseH mediated cleavage of its target RNA in cells or (e.g., as a mixmer) so as to inhibit translation of the target mRNA in cells. Antisense oligonucleotides for use in the instant disclosure may be modified in any suitable manner known in the art including, for example, as shown in U.S. Pat. No. 9,567,587, which is incorporated by reference herein for its disclosure regarding modification of antisense oligonucleotides (including, e.g., length, sugar moieties of the nucleobase (pyrimidine, purine), and alterations of the heterocyclic portion of the nucleobase). Further, antisense molecules have been used for decades to reduce expression of specific target genes (see, e.g., Bennett et al.; Pharmacology of Antisense Drugs, Annual Review of Pharmacology and Toxicology, Vol. 57: 81-105).

iv. Oligonucleotide Modifications

Oligonucleotides may be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation, immunogenicity, base-paring properties, RNA distribution and cellular uptake and other features relevant to therapeutic or research use. See, e.g., Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881; Bramsen and Kjems (Frontiers in Genetics, 3 (2012): 1-22). Accordingly, in some embodiments, oligonucleotides of the present disclosure may include one or more suitable modifications. In some embodiments, a modified nucleotide has a modification in its base (or nucleobase), the sugar (e.g., ribose, deoxyribose), or the phosphate group.

The number of modifications on an oligonucleotide and the positions of those nucleotide modifications may influence the properties of an oligonucleotide. For example, oligonucleotides may be delivered in vivo by conjugating them to or encompassing them in a lipid nanoparticle (LNP) or similar carrier. However, when an oligonucleotide is not protected by an LNP or similar carrier (e.g., “naked delivery”), it may be advantageous for at least some of the its nucleotides to be modified. Accordingly, in certain embodiments of any of the oligonucleotides provided herein, all or substantially all of the nucleotides of an oligonucleotide are modified. In certain embodiments, more than half of the nucleotides are modified. In certain embodiments, less than half of the nucleotides are modified. Typically, with naked delivery, every nucleotide is modified at the 2′-position of the sugar group of that nucleotide. These modifications may be reversible or irreversible. Typically, the 2′-position modification is 2′-fluoro, 2′-O-methyl, etc. In some embodiments, an oligonucleotide as disclosed herein has a number and type of modified nucleotides sufficient to cause the desired characteristic (e.g., protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and/or thermodynamic stability).

a. Sugar Modifications

In some embodiments, a modified sugar (also referred to herein as a sugar analog) includes a modified deoxyribose or ribose moiety, e.g., in which one or more modifications occur at the 2′, 3′, 4′, and/or 5′ carbon position of the sugar. In some embodiments, a modified sugar may also include non-natural alternative carbon structures such as those present in locked nucleic acids (“LNA”) (see, e.g., Koshkin et al. (1998), Tetrahedron 54, 3607-3630), unlocked nucleic acids (“UNA”) (see, e.g., Snead et al. (2013), Molecular Therapy—Nucleic Acids, 2, e103), and bridged nucleic acids (“BNA”) (see, e.g., Imanishi and Obika (2002), The Royal Society of Chemistry, Chem. Commun., 1653-1659). Koshkin et al., Snead et al., and Imanishi and Obika are incorporated by reference herein for their disclosures relating to sugar modifications.

In some embodiments, a nucleotide modification in a sugar comprises a 2′-modification. In certain embodiments, the 2′-modification may be 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, or 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid. Typically, the modification is 2′-fluoro, 2′-O-methyl, or 2′-O-methoxyethyl. However, a large variety of 2′ position modifications that have been developed for use in oligonucleotides can be employed in oligonucleotides disclosed herein. See, e.g., Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881. In some embodiments, a modification in a sugar comprises a modification of the sugar ring, which may comprise modification of one or more carbons of the sugar ring. For example, a modification of a sugar of a nucleotide may comprise a linkage between the 2′-carbon and a 1′-carbon or 4′-carbon of the sugar. For example, the linkage may comprise an ethylene or methylene bridge. In some embodiments, a modified nucleotide has an acyclic sugar that lacks a 2′-carbon to 3′-carbon bond. In some embodiments, a modified nucleotide has a thiol group, e.g., in the 4′ position of the sugar.

In some embodiments, the terminal 3′-end group (e.g., a 3′-hydroxyl) is a phosphate group or other group, which can be used, for example, to attach linkers, adapters or labels or for the direct ligation of an oligonucleotide to another nucleic acid.

b. 5′ Terminal Phosphates

5′-terminal phosphate groups of oligonucleotides may or in some circumstances enhance the interaction with Argonaut 2. However, oligonucleotides comprising a 5′-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo. In some embodiments, oligonucleotides include analogs of 5′ phosphates that are resistant to such degradation. In some embodiments, a phosphate analog may be oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 5′ end of an oligonucleotide strand is attached to a chemical moiety that mimics the electrostatic and steric properties of a natural 5′-phosphate group (“phosphate mimic”) (see, e.g., Prakash et al. (2015), Nucleic Acids Res., Nucleic Acids Res. 2015 Mar. 31; 43(6): 2993-3011, the contents of which relating to phosphate analogs are incorporated herein by reference). Many phosphate mimics have been developed that can be attached to the 5′ end (see, e.g., U.S. Pat. No. 8,927,513, the contents of which relating to phosphate analogs are incorporated herein by reference). Other modifications have been developed for the 5′ end of oligonucleotides (see, e.g., WO 2011/133871, the contents of which relating to phosphate analogs are incorporated herein by reference). In certain embodiments, a hydroxyl group is attached to the 5′ end of the oligonucleotide.

In some embodiments, an oligonucleotide has a phosphate analog at a 4′-carbon position of the sugar (referred to as a “4′-phosphate analog”). See, for example, International Patent Application PCT/US2017/049909, filed on Sep. 1, 2017, U.S. Provisional Application Nos. 62/383,207, entitled 4′-Phosphate Analogs and Oligonucleotides Comprising the Same, filed on Sep. 2, 2016, and 62/393,401, filed on Sep. 12, 2016, entitled 4′-Phosphate Analogs and Oligonucleotides Comprising the Same, the contents of each of which relating to phosphate analogs are incorporated herein by reference. In some embodiments, an oligonucleotide provided herein comprises a 4′-phosphate analog at a 5′-terminal nucleotide. In some embodiments, a phosphate analog is an oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof. In other embodiments, a 4′-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bound to the 4′-carbon of the sugar moiety or analog thereof. In certain embodiments, a 4′-phosphate analog is an oxymethylphosphonate. In some embodiments, an oxymethylphosphonate is represented by the formula —O—CH₂—PO(OH)₂ or —O—CH₂—PO(OR)₂, in which R is independently selected from H, CH₃, an alkyl group, CH₂CH₂CN, CH₂OCOC(CH₃)₃, CH₂OCH₂CH₂Si(CH₃)₃, or a protecting group. In certain embodiments, the alkyl group is CH₂CH₃. More typically, R is independently selected from H, CH₃, or CH₂CH₃.

c. Modified Internucleoside Linkages

In some embodiments, the oligonucleotide may comprise a modified internucleoside linkage. In some embodiments, phosphate modifications or substitutions may result in an oligonucleotide that comprises at least one (e.g., at least 1, at least 2, at least 3, at least 4, or at least 5) modified internucleotide linkage. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1 to 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3 or 1 to 2) modified internucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

A modified internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage or a boranophosphate linkage. In some embodiments, at least one modified internucleotide linkage of any one of the oligonucleotides as disclosed herein is a phosphorothioate linkage.

d. Base modifications

In some embodiments, oligonucleotides provided herein have one or more modified nucleobases. In some embodiments, modified nucleobases (also referred to herein as base analogs) are linked at the 1′ position of a nucleotide sugar moiety. In certain embodiments, a modified nucleobase is a nitrogenous base. In certain embodiments, a modified nucleobase does not contain a nitrogen atom. See e.g., U.S. Published Patent Application No. 20080274462. In some embodiments, a modified nucleotide comprises a universal base. However, in certain embodiments, a modified nucleotide does not contain a nucleobase (abasic).

In some embodiments, a universal base is a heterocyclic moiety located at the 1′ position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution that, when present in a duplex, can be positioned opposite more than one type of base without substantially altering the structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., oligonucleotide) that is fully complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower T_m than a duplex formed with the complementary nucleic acid. However, in some embodiments, compared to a reference single-stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher T_m than a duplex formed with the nucleic acid comprising the mismatched base.

Non-limiting examples of universal-binding nucleotides include inosine, 1-β-D-ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362 to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov. 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul. 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue. Nucleic Acids Res. 1994 Oct. 11; 22(20):4039-43. Each of the foregoing is incorporated by reference herein for their disclosures relating to base modifications).

e. Reversible Modifications

While certain modifications to protect an oligonucleotide from the in vivo environment before reaching target cells can be made, they can reduce the potency or activity of the oligonucleotide once it reaches the cytosol of the target cell. Reversible modifications can be made such that the molecule retains desirable properties outside of the cell, which are then removed upon entering the cytosolic environment of the cell. Reversible modification can be removed, for example, by the action of an intracellular enzyme or by the chemical conditions inside of a cell (e.g., through reduction by intracellular glutathione).

In some embodiments, a reversibly modified nucleotide comprises a glutathione-sensitive moiety. Typically, nucleic acid molecules have been chemically modified with cyclic disulfide moieties to mask the negative charge created by the internucleotide diphosphate linkages and improve cellular uptake and nuclease resistance. See U.S. Published Application No. 2011/0294869 originally assigned to Traversa Therapeutics, Inc. (“Traversa”), PCT Publication No. WO 2015/188197 to Solstice Biologics, Ltd. (“Solstice”), Meade et al., Nature Biotechnology, 2014, 32:1256-1263 (“Meade”), PCT Publication No. WO 2014/088920 to Merck Sharp & Dohme Corp, each of which are incorporated by reference for their disclosures of such modifications. This reversible modification of the internucleotide diphosphate linkages is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g. glutathione). Earlier examples include neutralizing phosphotriester modifications that were reported to be cleavable inside cells (Dellinger et al. J. Am. Chem. Soc. 2003, 125:940-950).

In some embodiments, such a reversible modification allows protection during in vivo administration (e.g., transit through the blood and/or lysosomal/endosomal compartments of a cell) where the oligonucleotide will be exposed to nucleases and other harsh environmental conditions (e.g., pH). When released into the cytosol of a cell where the levels of glutathione are higher compared to extracellular space, the modification is reversed and the result is a cleaved oligonucleotide. Using reversible, glutathione sensitive moieties, it is possible to introduce sterically larger chemical groups into the oligonucleotide of interest as compared to the options available using irreversible chemical modifications. This is because these larger chemical groups will be removed in the cytosol and, therefore, should not interfere with the biological activity of the oligonucleotides inside the cytosol of a cell. As a result, these larger chemical groups can be engineered to confer various advantages to the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the glutathione-sensitive moiety can be engineered to modify the kinetics of its release.

In some embodiments, a glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, a glutathione-sensitive moiety is attached to the 2′-carbon of the sugar of a modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5′-carbon of a sugar, particularly when the modified nucleotide is the 5′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3′-carbon of a sugar, particularly when the modified nucleotide is the 3′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, e.g., International Patent Application PCT/US2017/048239, which published on Mar. 1, 2018 as International Patent Publication WO2018/039364, entitled Compositions Comprising Reversibly Modified Oligonucleotides and Uses Thereof, which was filed on Aug. 23, 2016, the contents of which are incorporated by reference herein for its relevant disclosures.

v. Targeting Ligands

In some embodiments, it may be desirable to target the oligonucleotides of the disclosure to one or more cells or one or more organs. Such a strategy may help to avoid undesirable effects in other organs, or may avoid undue loss of the oligonucleotide to cells, tissue or organs that would not benefit for the oligonucleotide. Accordingly, in some embodiments, oligonucleotides disclosed herein may be modified to facilitate targeting of a particular tissue, cell or organ, e.g., to facilitate delivery of the oligonucleotide to the liver. In certain embodiments, oligonucleotides disclosed herein may be modified to facilitate delivery of the oligonucleotide to the hepatocytes of the liver. In some embodiments, an oligonucleotide comprises a nucleotide that is conjugated to one or more targeting ligands.

A targeting ligand may comprise a carbohydrate, amino sugar, cholesterol, peptide, polypeptide, protein or part of a protein (e.g., an antibody or antibody fragment) or lipid. In some embodiments, a targeting ligand is an aptamer. For example, a targeting ligand may be an RGD peptide that is used to target tumor vasculature or glioma cells, CREKA peptide to target tumor vasculature or stoma, transferrin, lactoferrin, or an aptamer to target transferrin receptors expressed on CNS vasculature, or an anti-EGFR antibody to target EGFR on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties.

In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5′ or 3′ end of the sense or antisense strand) such that the targeting ligands resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may comprise a stem-loop at either the 5′ or 3′ end of the sense strand and 1, 2, 3, or 4 nucleotides of the loop of the stem may be individually conjugated to a targeting ligand, as described, for example, in International Patent Application Publication WO 2016/100401, which was published on Jun. 23, 2016, the relevant contents of which are incorporated herein by reference.

In some embodiments, it is desirable to target an oligonucleotide that reduces the expression of GYS2 to the hepatocytes of the liver of a subject. Any suitable hepatocyte targeting moiety may be used for this purpose.

GalNAc is a high affinity ligand for asialoglycoprotein receptor (ASGPR), which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to oligonucleotides of the instant disclosure may be used to target these oligonucleotides to the ASGPR expressed on these hepatocyte cells.

In some embodiments, an oligonucleotide of the instant disclosure is conjugated directly or indirectly to a monovalent GalNAc. In some embodiments, the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc (i.e., is conjugated to 2, 3, or 4 monovalent GalNAc moieties, and is typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, an oligonucleotide of the instant disclosure is conjugated to one or more bivalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties.

In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of the loop (L) of the stem-loop are each conjugated to a separate GalNAc. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5′ or 3′ end of the sense or antisense strand) such that the GalNAc moieties resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may comprise a stem-loop at either the 5′ or 3′ end of the sense strand and 1, 2, 3, or 4 nucleotides of the loop of the stem may be individually conjugated to a GalNAc moiety. In some embodiments, GalNAc moieties are conjugated to a nucleotide of the sense strand. For example, four GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand, where each GalNAc moiety is conjugated to one nucleotide.

Appropriate methods or chemistry (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication Number WO2016100401 A1, which published on Jun. 23, 2016, and the contents of which relating to such linkers are incorporated herein by reference. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is fairly stable. In some embodiments, a duplex extension (up to 3, 4, 5, or 6 base pairs in length) is provided between a targeting ligand (e.g., a GalNAc moiety) and a double-stranded oligonucleotide.

III. Formulations

Various formulations have been developed to facilitate oligonucleotide use. For example, oligonucleotides can be delivered to a subject or a cellular environment using a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the oligonucleotides in the formulation. In some embodiments, provided herein are compositions comprising oligonucleotides (e.g., single-stranded or double-stranded oligonucleotides) to reduce the expression of GYS2. Such compositions can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient portion of the oligonucleotides enter the cell to reduce GYS2 expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for the reduction of GYS2 as disclosed herein. In some embodiments, an oligonucleotide is formulated in buffer solutions such as phosphate-buffered saline solutions, liposomes, micellar structures, and capsids. In some embodiments, naked oligonucleotides or conjugates thereof are formulated in water or in an aqueous solution (e.g., water with pH adjustments). In some embodiments, naked oligonucleotides or conjugates thereof are formulated in basic buffered aqueous solutions (e.g., PBS)

Formulations of oligonucleotides with cationic lipids can be used to facilitate transfection of the oligonucleotides into cells. For example, cationic lipids, such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be used according to the manufacturer's instructions.

Accordingly, in some embodiments, a formulation comprises a lipid nanoparticle. In some embodiments, an excipient comprises a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof (see, e.g., Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2013).

In some embodiments, formulations as disclosed herein comprise an excipient. In some embodiments, an excipient confers to a composition improved stability, improved absorption, improved solubility and/or therapeutic enhancement of the active ingredient. In some embodiments, an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, an oligonucleotide is lyophilized for extending its shelf-life and then made into a solution before use (e.g., administration to a subject). Accordingly, an excipient in a composition comprising any one of the oligonucleotides described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone), or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Typically, the route of administration is intravenous or subcutaneous.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the oligonucleotides in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

In some embodiments, a composition may contain at least about 0.1% of the therapeutic agent (e.g., an oligonucleotide for reducing GYS2 expression) or more, although the percentage of the active ingredient(s) may be between about 1% and about 80% or more of the weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

Even though a number of embodiments are directed to liver-targeted delivery of any of the oligonucleotides disclosed herein, targeting of other tissues is also contemplated.

IV. Methods of Use

i. Reducing GYS2 Expression in Cells

In some embodiments, methods are provided for delivering to a cell an effective amount any one of oligonucleotides disclosed herein for purposes of reducing expression of GYS2 in the cell. Methods provided herein are useful in any appropriate cell type. In some embodiments, a cell is any cell that expresses GYS2 (e.g., liver cells such as hepatocytes or adipose cells). In some embodiments, the cell is a primary cell that has been obtained from a subject and that may have undergone a limited number of a passages, such that the cell substantially maintains its natural phenotypic properties. In some embodiments, a cell to which the oligonucleotide is delivered is ex vivo or in vitro (i.e., can be delivered to a cell in culture or to an organism in which the cell resides). In specific embodiments, methods are provided for delivering to a cell an effective amount any one of the oligonucleotides disclosed herein for purposes of reducing expression of GYS2 solely or primarily in hepatocytes.

In some embodiments, oligonucleotides disclosed herein can be introduced using appropriate nucleic acid delivery methods including injection of a solution containing the oligonucleotides, bombardment by particles covered by the oligonucleotides, exposing the cell or organism to a solution containing the oligonucleotides, or electroporation of cell membranes in the presence of the oligonucleotides. Other appropriate methods for delivering oligonucleotides to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and others.

The consequences of inhibition can be confirmed by an appropriate assay to evaluate one or more properties of a cell or subject, or by biochemical techniques that evaluate molecules indicative of GYS2 expression (e.g., RNA, protein). In some embodiments, the extent to which an oligonucleotide provided herein reduces levels of expression of GYS2 is evaluated by comparing expression levels (e.g., mRNA or protein levels of GYS2 to an appropriate control (e.g., a level of GYS2 expression in a cell or population of cells to which an oligonucleotide has not been delivered or to which a negative control has been delivered). In some embodiments, an appropriate control level of GYS2 expression may be a predetermined level or value, such that a control level need not be measured every time. The predetermined level or value can take a variety of forms. In some embodiments, a predetermined level or value can be single cut-off value, such as a median or mean.

In some embodiments, administration of an oligonucleotide as described herein results in a reduction in the level of GYS2 expression in a cell. In some embodiments, the reduction in levels of GYS2 expression may be a reduction to 1% or lower, 5% or lower, 10% or lower, 15% or lower, 20% or lower, 25% or lower, 30% or lower, 35% or lower, 40% or lower, 45% or lower, 50% or lower, 55% or lower, 60% or lower, 70% or lower, 80% or lower, or 90% or lower compared with an appropriate control level of GYS2. The appropriate control level may be a level of GYS2 expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein. In some embodiments, the effect of delivery of an oligonucleotide to a cell according to a method disclosed herein is assessed after a finite period of time. For example, levels of GYS2 may be analyzed in a cell at least 8 hours, 12 hours, 18 hours, 24 hours; or at least one, two, three, four, five, six, seven, or fourteen days after introduction of the oligonucleotide into the cell.

In some embodiments, an oligonucleotide is delivered in the form of a transgene that is engineered to express in a cell the oligonucleotides (e.g., its sense and antisense strands). In some embodiments, an oligonucleotide is delivered using a transgene that is engineered to express any oligonucleotide disclosed herein. Transgenes may be delivered using viral vectors (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus or herpes simplex virus) or non-viral vectors (e.g., plasmids or synthetic mRNAs). In some embodiments, transgenes can be injected directly to a subject.

ii. Treatment Methods

Aspects of the disclosure relate to methods for reducing GYS2 expression for the treatment of a glycogen storage disease in a subject. In some embodiments, the methods may comprise administering to a subject in need thereof an effective amount of any one of the oligonucleotides disclosed herein. Such treatments could be used, for example, to decrease or prevent hepatomegaly, liver toxicity (e.g., lower or decrease levels of AST, ALT, and/or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma. Such treatments could also be used, for example, to treat or prevent one or more symptoms associated with a glycogen storage disease selected from the list consisting of: GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX, or to treat or prevent one or more symptoms of such a glycogen storage disease. The present disclosure provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) and/or symptoms or conditions associated with a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX).

In certain aspects, the disclosure provides a method for preventing in a subject, a disease, disorder, symptom, or condition as described herein by administering to the subject a therapeutic agent (e.g., an oligonucleotide or vector or transgene encoding same). In some embodiments, the subject to be treated is a subject who will benefit therapeutically from a reduction in the amount of GYS2 protein, e.g., in the liver.

Methods described herein typically involve administering to a subject an effective amount of an oligonucleotide, that is, an amount capable of producing a desirable therapeutic result. A therapeutically acceptable amount may be an amount that is capable of treating a disease or disorder. The appropriate dosage for any one subject will depend on certain factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently.

In some embodiments, a subject is administered any one of the compositions disclosed herein either enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intramuscular injection,), topically (e.g., epicutaneous, inhalational, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the liver of a subject). Typically, oligonucleotides disclosed herein are administered intravenously or subcutaneously.

In some embodiments, oligonucleotides are administered at a dose in a range of 0.1 mg/kg to 25 mg/kg (e.g., 1 mg/kg to 5 mg/kg). In some embodiments, oligonucleotides are administered at a dose in a range of 0.1 mg/kg to 5 mg/kg or in a range of 0.5 mg/kg to 5 mg/kg.

As a non-limiting set of examples, the oligonucleotides of the instant disclosure would typically be administered once per year, twice per year, quarterly (once every three months), bi-monthly (once every two months), monthly, or weekly.

In some embodiments, the subject to be treated is a human (e.g., a human patient) or non-human primate or other mammalian subject. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

EXAMPLES

Example 1: Development of GYS2 Oligonucleotide Inhibitors Using Human and Mouse Cell-Based Assays

Human and mouse-based assays were used to develop candidate oligonucleotides for inhibition of GYS2 expression. First, a computer-based algorithm was used to generate candidate oligonucleotide sequences (25-27-mer) for GYS2 inhibition. Cell-based assays and PCR assays were then employed for evaluation of candidate oligonucleotides for their ability to reduce GYS2 expression.

The computer-based algorithm provided oligonucleotides that were complementary to the human GYS2 mRNA (SEQ ID NO: 609, Table 1), of which certain sequences were also complementary to the Rhesus macaque GYS2 mRNA (SEQ ID NO: 610, Table 1).

TABLE 1
Sequences of human and Rhesus macaque GYS2 mRNA
	Species	GenBank RefSeq #	SEQ ID NO.
	Human	NM_021957.3	609
	Rhesus macaque	XM_001098578.2	610

Of the oligonucleotides that the algorithm provided, 264 oligonucleotides were selected as candidates for experimental evaluation in a HEK-293 cell-based assay. In this assay, HEK-293 human embryonic kidney cells stably expressing GYS2 (referred to as HEK-GYS2 cells) were transfected with the oligonucleotides. Cells were maintained for a period of time following transfection and then levels of remaining GYS2 mRNA were interrogated using TAQMAN®-based qPCR assays. Two qPCR assays, a 3′ assay and a 5′ assay, were used to determine mRNA levels as measured by HEX and FAM probes, respectively. The results of the HEK-293 cell-based assay with the 264 oligonucleotides are shown in FIGS. 1A and 1B. The percent mRNA remaining is shown for each of the 3′ assay (circle shapes) and the 5′ assay (diamond shapes). Oligonucleotides with the lowest percentage of mRNA remaining compared to negative controls were considered hits. Oligonucleotides with low complementarity to the human genome were used as negative controls.

Based on the activity and locations of these oligonucleotides, hotspots on the human GYS2 mRNA were defined. A hotspot was identified as a stretch on the human GYS2 mRNA sequence associated with at least two oligonucleotides resulting in mRNA levels that were less than or equal to 35% in either assay compared with controls. Accordingly, the following hotspots within the human GYS2 mRNA sequence were identified: 579-618, 691-738, 1089-1125, 1175-1211, 1431-1486, 2341-2383, 2497-2543, 2660-2698, 2808-2851, and 3014-3050.

The sequences of the hotspots are outlined in Table 2.

TABLE 2
Sequences of Hotspots
Hotspot
Position
In Human		SEQ
GYS2		ID
mRNA	Sequence	NO.
579-618	GATAGAAGGAAGTCCTTATGTGGTACTTTTTGACATA	599
	GGC
691-738	GACCGAGAAGCCAATGATATGCTGATATTTGGATCTT	600
	TAACTGCCTGG
1089-1125	TCCAAACGGCTTGAATGTTAAGAAATTTTCAGCAGTG	601
1175-1211	TTGTTCGAGGTCATTTCTATGGICATCTCGACTTTGA	602
1431-1486	TGCACATTCTGTGAAGGAAAAGTTTGGAAAAAAACTC	603
	TATGATGCATTATTAAGAG
2341-2383	AAGCTGCATGGTGAATATAAGAACTGAATTCTACATG	604
	TGCTGC
2497-2543	GTGGAAGAAATTGAGTGAATGACAATTTTGTAATTTA	605
	GGATAAGATC
2660-2698	TTTCTCTTACTCTGTTTATTTTTAAATGATCATCATA	606
	AT
2808-2851	TAGCTAGGTTTTTACTGATTATTTTCATTTTTCACAT	607
	GCATCAG
3014-3050	TCTTACTGTAACATTTTTCTATTGTTTAAATAGAAAG	608

Dose Response Analysis

Of the 264 oligonucleotides evaluated in the initial HEK-293 cell-based assay, 71 particularly active oligonucleotides were selected as hits based on their ability to knock down GYS2 levels and were subjected to a secondary screen.

In this secondary screen, the candidate oligonucleotides were tested using the same assay as in the primary screen, but at two or three different concentrations (1 nM, 0.1 nM and 0.03 nM) (FIGS. 2A and 2B). The target mRNA levels were generally normalized based on splicing factor, arginine/serine-rich 9 (SFRS9), a housekeeping gene that provides a stable expression reference across samples, to generate the percent mRNA shown in FIGS. 2A and 2B. The tested oligonucleotides in each of FIGS. 2A and 2B are shown compared to negative control sequences (NC1) and mock transfection. All 71 oligonucleotides had the same modification pattern, designated M1, which contains a combination of ribonucleotides, deoxyribonucleotides and 2′-O-methyl modified nucleotides. The sequences of the 71 oligonucleotides tested are provided in Table 3.

TABLE 3
Candidate oligonucleotide Sequences
for HEK-293 Cell-Based Assay
		Sense	Corresponding Antisense
Hs	Rm	SEQ ID NO.	SEQ ID NO.
X	X	1-3, 5, 6, 8, 9, 11-13,	193-195, 197, 198, 200, 201,
		15-17, 19, 21-24, 52-54,	203-205, 207-209, 211, 213-
		59, 62, 72, 73, 77, 82,	216, 244-246, 251, 254, 264,
		84-86, 89, 93-95, 98,	265, 269, 274, 276-278, 281,
		104, 106, 109, 111, 114,	285-287, 290, 296, 298, 301,
		116-118, 129, 141-144, 158,	303, 306, 308-310, 321, 333-
		160, 161, 177, 185, 186, 189,	336, 350, 352, 353, 369, 377,
		192, 468, 469, 472, 473,	378, 381, 384, 519, 520, 523,
		478-480, 482, 483, 487,	524, 529-531, 533, 534, 538,
		488, 490-492, 498	539, 541-543, 549
Hs: human and Rm: Rhesus macaque; the sense and antisense SEQ ID NO. columns provide the sense strand and respective antisense strand, in relative order, that are hybridized to make each oligonucleotide. For example, sense strand of SEQ ID NO: 1 hybridizes with antisense strand of SEQ ID NO: 193.

At this stage, 36 of the most potent sequences from the testing were selected for further analysis. The selected sequences were converted to nicked tetraloop structure formats (a 36-mer passenger strand with a 22-mer guide strand). See FIG. 3 for a generic tetraloop structure. These oligonucleotides were then tested as before, evaluating each oligonucleotide at three concentrations for its ability to reduce GYS2 mRNA expression in HepG2 cells.

FIG. 4 shows data for oligonucleotides made from different base sequences with nicked tetraloop structures, each adapted to one or two different modification patterns. The X-axis lists the 3′ end of the sense strand targeted by the oligonucleotide evaluated. The target mRNA levels were normalized as described above to generate the percent mRNA shown in FIG. 4, and the tested oligonucleotides are shown compared to negative control sequences (NC1) and mock transfection.

Certain tetraloop-modified oligonucleotides were further tested in monkey hepatocyte cells using the same modification patterns for each compound (FIG. 5) at 0.1 μM, 0.3 μM, and 1.0 μM. The tested oligonucleotides in FIG. 5 are shown compared untransfected cells. Certain oligonucleotides were further tested using a full dose response curve in HEK-293 cells in order to determine the half maximal inhibitory concentration (IC₅₀) for each compound (see FIGS. 6A and 6B).

In Vivo Murine Screening

Data from the above in vitro experiments were assessed to identify tetraloops and modification patterns that would improve delivery properties while maintaining activity for reduction of GYS2 expression in the mouse hepatocytes. Based on this analysis, select oligonucleotides were then conjugated to GalNAc moieties. Four GalNAc moieties were conjugated to nucleotides in the tetraloop of the sense strand. Conjugation was performed using a click linker. The GalNAc used was as shown below:

A total of 65 potent GalNAc-conjugated GYS2 oligonucleotides from 18 different base sequences and having different modification patterns with nicked tetraloop structures were tested. Selected GYS2 oligonucleotide sequences were active against human and monkey mRNA sequences but not mouse Gys2. GYS2 oligonucleotides were subcutaneously administered to CD-1 mice transiently expressing human GYS2 mRNA by hydrodynamic injection of a human GYS2 expression plasmid at 0.5-5 mg/kg. Mice were euthanized on day 4 following administration. Liver samples were obtained and RNA was extracted to evaluate GYS2 mRNA levels by RT-qPCR. The percent GYS2 mRNA as compared to PBS control mRNA was determined based on these measurements.

From the 65 conjugates tested, the eight most potent base sequences were identified and each tested with the same modification pattern by subcutaneous injection at 0.5 mg/kg to CD-1 mice transiently expressing human GYS2 mRNA. Mice were euthanized on day 4 following administration. Liver samples were obtained and RNA was extracted to evaluate GYS2 mRNA levels by RT-qPCR. The percent GYS2 mRNA as compared to PBS control mRNA was determined based on these measurements and is shown in FIG. 7.

Materials and Methods

Transfection

For the first screen, Lipofectamine RNAiMAX™ was used to complex the oligonucleotides for efficient transfection. Oligonucleotides, RNAiMAX and Opti-MEM incubated together at room temperature for 20 minutes and then 50 μL of this mix was added per well to plates prior to transfection. Media was aspirated from a flask of actively passaging cells and the cells were incubated at 37° C. in the presence of trypsin for 3-5 minutes. After cells no longer adhered to the flask, cell growth media (lacking penicillin and streptomycin) was added to neutralize the trypsin and to suspend the cells. A 10 μL aliquot was removed and counted with a hemocytometer to quantify the cells on a per milliliter basis. For cells, 10,000 or 25,000 cells/well were seeded per well in media (e.g., 100 μL of media). A diluted cell suspension was added to the 96-well transfection plates, which already contained the oligonucleotides in Opti-MEM. The transfection plates were then incubated for 24 hours at 37° C. After 24 hours of incubation, media was aspirated from each well. Cells were lysed using the lysis buffer from the Promega RNA Isolation kit. The lysis buffer was added to each well. The lysed cells were then transferred to the Corbett XtractorGENE (QIAxtractor) for RNA isolation or stored at −80° C.

For subsequent screens and experiments, e.g., the secondary screen, Lipofectamine RNAiMAx was used to complex the oligonucleotides for reverse transfection. The complexes were made by mixing RNAiMAX and siRNAs in OptiMEM medium for 15 minutes. The transfection mixture was transferred to multi-well plates and cell suspension was added to the wells. After 24 hours incubation the cells were washed once with PBS and then lysed using lysis buffer from the Promega SV96 kit. The RNA was purified using the SV96 plates in a vacuum manifold. Four microliters of the purified RNA was then heated at 65° C. for 5 minutes and cooled to 4° C. The RNA was then used for reverse transcription using the High Capacity Reverse Transcription kit (Life Technologies) in a 10 microliter reaction. The cDNA was then diluted to 50 μL with nuclease free water and used for quantitative PCR with multiplexed 5′-endonuclease assays and SSoFast qPCR mastermix (Bio-Rad laboratories).

cDNA Synthesis

RNA was isolated from mammalian cells in tissue culture using the Corbett X-tractor Gene™ (QIAxtractor). A modified SuperScript II protocol was used to synthesize cDNA from the isolated RNA. Isolated RNA (approximately 5 ng/μL) was heated to 65° C. for five minutes and incubated with dNPs, random hexamers, oligo dTs, and water. The mixture was cooled for 15 seconds. An “enzyme mix,” consisting of water, 5× first strand buffer, DTT, SUPERase⋅In™ (an RNA inhibitor), and SuperScript II RTase was added to the mixture. The contents were heated to 42° C. for one hour, then to 70° C. for 15 minutes, and then cooled to 4° C. using a thermocycler. The resulting cDNA was then subjected to SYBR®-based qPCR. The qPCR reactions were multiplexed, containing two 5′ endonuclease assays per reaction.

qPCR Assays

Primer sets were initially screened using SYBR®-based qPCR. Assay specificity was verified by assessing melt curves as well as “minus RT” controls. Dilutions of cDNA template (10-fold serial dilutions from 20 ng and to 0.02 ng per reaction) from HeLa and Hepa1-6 cells were used to test human (Hs) and mouse (Mm) assays, respectively. qPCR assays were set up in 384-well plates, covered with MicroAmp film, and run on the 7900HT from Applied Biosystems. Reagent concentrations and cycling conditions included the following: 2×SYBR mix, 10 μM forward primer, 10 μM reverse primer, DD H₂O, and cDNA template up to a total volume of 10 μL.

Cloning

PCR amplicons that displayed a single melt-curve were ligated into the pGEM®-T Easy vector kit from Promega according to the manufacturer's instructions. Following the manufacturer's protocol, JM109 High Efficiency cells were transformed with the newly ligated vectors. The cells were then plated on LB plates containing ampicillin and incubated at 37° C. overnight for colony growth.

PCR Screening and Plasmid Mini-Prep

PCR was used to identify colonies of E. coli that had been transformed with a vector containing the ligated amplicon of interest. Vector-specific primers that flank the insert were used in the PCR reaction. All PCR products were then run on a 1% agarose gel and imaged by a transilluminator following staining. Gels were assessed qualitatively to determine which plasmids appeared to contain a ligated amplicon of the expected size (approximately 300 bp, including the amplicon and the flanking vector sequences specific to the primers used).

The colonies that were confirmed transformants by PCR screening were then incubated overnight in cultures consisting of 2 mL LB broth with ampicillin at 37° C. with shaking. E. coli cells were then lysed, and the plasmids of interest were isolated using Promega's Mini-Prep kit. Plasmid concentration was determined by UV absorbance at 260 nm.

Plasmid Sequencing and Quantification

Purified plasmids were sequenced using the BigDye® Terminator sequencing kit. The vector-specific primer, T7, was used to give read lengths that span the insert. The following reagents were used in the sequencing reactions: water, 5× sequencing buffer, BigDye terminator mix, T7 primer, and plasmid (100 ng/μL) to a volume of 10 μL. The mixture was held at 96° C. for one minute, then subjected to 15 cycles of 96° C. for 10 seconds, 50° C. for 5 seconds, 60° C. for 1 minute, 15 seconds; 5 cycles of 96° C. for 10 seconds, 50° C. for 5 seconds, 60° C. for 1 minute, 30 seconds; and 5 cycles of 96° C. for 10 seconds, 50° C. for 5 seconds, and 60° C. for 2 minutes. Dye termination reactions were then sequenced using Applied Biosystems' capillary electrophoresis sequencers.

Sequence-verified plasmids were then quantified. They were linearized using a single cutting restriction endonuclease. Linearity was confirmed using agarose gel electrophoresis. All plasmid dilutions were made in TE buffer (pH 7.5) with 100 μg of tRNA per mL buffer to reduce non-specific binding of plasmid to the polypropylene vials.

The linearized plasmids were then serially diluted from 1,000,000 to 01 copies per μL and subjected to qPCR. Assay efficiency was calculated and the assays were deemed acceptable if the efficiency was in the range of 90-110%.

Multi-Plexing Assays

For each target, mRNA levels were quantified by two 5′ nuclease assays. In general, several assays are screened for each target. The two assays selected displayed a combination of good efficiency, low limit of detection, and broad 5′→3′ coverage of the gene of interest (GOI). Both assays against one GOI could be combined in one reaction when different fluorophores were used on the respective probes. Thus, the final step in assay validation was to determine the efficiency of the selected assays when they were combined in the same qPCR or “multi-plexed.”

Linearized plasmids for both assays in 10-fold dilutions were combined and qPCR was performed. The efficiency of each assay was determined as described above. The accepted efficiency rate was 90-110%. While validating multi-plexed reactions using linearized plasmid standards, C_q values for the target of interest were also assessed using cDNA as the template. For human or mouse targets, HeLa and Hepa1-6 cDNA were used, respectively. The cDNA, in this case, was derived from RNA isolated on the Corbett (˜5 ng/μl in water) from untransfected cells. In this way, the observed C_q values from this sample cDNA were representative of the expected C_q values from a 96-well plate transfection. In cases where C_q values were greater than 30, other cell lines were sought that exhibit higher expression levels of the gene of interest. A library of total RNA isolated from via high-throughput methods on the Corbett from each human and mouse line was generated and used to screen for acceptable levels of target expression.

Description of Oligonucleotide Nomenclature

All oligonucleotides described herein are designated either SN₁-ASN₂-MN₃. The following designations apply:

• • N₁: sequence identifier number of the sense strand sequence
  • N₂: sequence identifier number of the antisense strand sequence
  • N₃: reference number of modification pattern, in which each number represents a pattern of modified nucleotides in the oligonucleotide.
    For example, S27-AS219-M1 represents an oligonucleotide with a sense sequence that is set forth by SEQ ID NO: 27, an antisense sequence that is set forth by SEQ ID NO: 219, and which is adapted to a modification pattern identified as M1.

TABLE 4
GYS2 RNAi oligonucleotides
		S SEQ		AS SEQ
App Name	Sense Sequence	ID NO	Antisense Sequence	ID NO
S1-AS193-	CAGGUGCAUUUUGGAAGAUGGCUGA	1	UCAGCCAUCUUCCAAAAUG	193
M1			CACCUGGC
S2-AS194-	AGGUGCAUUUUGGAAGAUGGCUGAT	2	AUCAGCCAUCUUCCAAAAU	194
M1			GCACCUGG
S3-AS195-	GGUGCAUUUUGGAAGAUGGCUGATA	3	UAUCAGCCAUCUUCCAAAA	195
M1			UGCACCUG
S4-AS196-	GUGCAUUUUGGAAGAUGGCUGAUAG	4	CUAUCAGCCAUCUUCCAAA	196
M1			AUGCACCU
S5-AS197-	UGCAUUUUGGAAGAUGGCUGAUAGA	5	UCUAUCAGCCAUCUUCCAA	197
M1			AAUGCACC
S6-AS198-	AAGGAAGUCCUUAUGUGGUACUUTT	6	AAAAGUACCACAUAAGGAC	198
M1			UUCCUUCU
S7-AS199-	GAAGUCCUUAUGUGGUACUUUUUGA	7	UCAAAAAGUACCACAUAAG	199
M1			GACUUCCU
S8-AS200-	AAGUCCUUAUGUGGUACUUUUUGAC	8	GUCAAAAAGUACCACAUAA	200
M1			GGACUUCC
S9-AS201-	AGUCCUUAUGUGGUACUUUUUGACA	9	UGUCAAAAAGUACCACAUA	201
M1			AGGACUUC
S10-AS202-	GUCCUUAUGUGGUACUUUUUGACAT	10	AUGUCAAAAAGUACCACAU	202
M1			AAGGACUU
S11-AS203-	CCUUAUGUGGUACUUUUUGACAUAG	11	CUAUGUCAAAAAGUACCAC	203
M1			AUAAGGAC
S12-AS204-	GUACUUUUUGACAUAGGCUAUUCAG	12	CUGAAUAGCCUAUGUCAAA	204
M1			AAGUACCA
S13-AS205-	CGAGAAGCCAAUGAUAUGCUGAUAT	13	AUAUCAGCAUAUCAUUGG	205
M1			CUUCUCGGU
S14-AS206-	GAGAAGCCAAUGAUAUGCUGAUATT	14	AAUAUCAGCAUAUCAUUG	206
M1			GCUUCUCGG
S15-AS207-	AGAAGCCAAUGAUAUGCUGAUAUTT	15	AAAUAUCAGCAUAUCAUU	207
M1			GGCUUCUCG
S16-AS208-	GAAGCCAAUGAUAUGCUGAUAUUTG	16	CAAAUAUCAGCAUAUCAUU	208
M1			GGCUUCUC
S17-AS209-	AGCCAAUGAUAUGCUGAUAUUUGGA	17	UCCAAAUAUCAGCAUAUCA	209
M1			UUGGCUUC
S18-AS210-	GCCAAUGAUAUGCUGAUAUUUGGAT	18	AUCCAAAUAUCAGCAUAUC	210
M1			AUUGGCUU
S19-AS211-	CCAAUGAUAUGCUGAUAUUUGGATC	19	GAUCCAAAUAUCAGCAUAU	211
M1			CAUUGGCU
S20-AS212-	AAUGAUAUGCUGAUAUUUGGAUCTT	20	AAGAUCCAAAUAUCAGCAU	212
M1			AUCAUUGG
S21-AS213-	AUGAUAUGCUGAUAUUUGGAUCUTT	21	AAAGAUCCAAAUAUCAGCA	213
M1			UAUCAUUG
S22-AS214-	UGAUAUGCUGAUAUUUGGAUCUUTA	22	UAAAGAUCCAAAUAUCAGC	214
M1			AUAUCAUU
S23-AS215-	AUAUGCUGAUAUUUGGAUCUUUAAC	23	GUUAAAGAUCCAAAUAUCA	215
M1			GCAUAUCA
S24-AS216-	AUGCUGAUAUUUGGAUCUUUAACTG	24	CAGUUAAAGAUCCAAAUAU	216
M1			CAGCAUAU
S25-AS217-	UCUUUAACUGCCUGGUUCUUAAAAG	25	CUUUUAAGAACCAGGCAG	217
M1			UUAAAGAUC
S26-AS218-	CUUUAACUGCCUGGUUCUUAAAAGA	26	UCUUUUAAGAACCAGGCA	218
M1			GUUAAAGAU
S27-AS219-	UUUAACUGCCUGGUUCUUAAAAGAG	27	CUCUUUUAAGAACCAGGCA	219
M1			GUUAAAGA
S28-AS220-	UUAACUGCCUGGUUCUUAAAAGAGG	28	CCUCUUUUAAGAACCAGGC	220
M1			AGUUAAAG
S29-AS221-	UAACUGCCUGGUUCUUAAAAGAGGT	29	ACCUCUUUUAAGAACCAGG	221
M1			CAGUUAAA
S30-AS222-	UUGCCCAAUUCCAUGAAUGGCAGGC	30	GCCUGCCAUUCAUGGAAU	222
M1			UGGGCAACG
S31-AS223-	UGCCCAAUUCCAUGAAUGGCAGGCT	31	AGCCUGCCAUUCAUGGAAU	223
M1			UGGGCAAC
S32-AS224-	GCCCAAUUCCAUGAAUGGCAGGCTG	32	CAGCCUGCCAUUCAUGGAA	224
M1			UUGGGCAA
S33-AS225-	CCCAAUUCCAUGAAUGGCAGGCUGG	33	CCAGCCUGCCAUUCAUGGA	225
M1			AUUGGGCA
S34-AS226-	CCAAUUCCAUGAAUGGCAGGCUGGA	34	UCCAGCCUGCCAUUCAUGG	226
M1			AAUUGGGC
S35-AS227-	CAAUUCCAUGAAUGGCAGGCUGGAA	35	UUCCAGCCUGCCAUUCAUG	227
M1			GAAUUGGG
S36-AS228-	AAUUCCAUGAAUGGCAGGCUGGAAT	36	AUUCCAGCCUGCCAUUCAU	228
M1			GGAAUUGG
S37-AS229-	AUUCCAUGAAUGGCAGGCUGGAATT	37	AAUUCCAGCCUGCCAUUCA	229
M1			UGGAAUUG
S38-AS230-	UUCCAUGAAUGGCAGGCUGGAAUTG	38	CAAUUCCAGCCUGCCAUUC	230
M1			AUGGAAUU
S39-AS231-	UCCAUGAAUGGCAGGCUGGAAUUGG	39	CCAAUUCCAGCCUGCCAUU	231
M1			CAUGGAAU
S40-AS232-	GGAAACUUCCUAUUGCCACAAUATT	40	AAUAUUGUGGCAAUAGGA	232
M1			AGUUUCCUG
S41-AS233-	GGUAUCUCUGUGCAGCAAAUAUUGA	41	UCAAUAUUUGCUGCACAG	233
M1			AGAUACCUC
S42-AS234-	GUAUCUCUGUGCAGCAAAUAUUGAT	42	AUCAAUAUUUGCUGCACA	234
M1			GAGAUACCU
S43-AS235-	UAUCUCUGUGCAGCAAAUAUUGATT	43	AAUCAAUAUUUGCUGCACA	235
M1			GAGAUACC
S44-AS236-	AUCUCUGUGCAGCAAAUAUUGAUTT	44	AAAUCAAUAUUUGCUGCAC	236
M1			AGAGAUAC
S45-AS237-	UCUCUGUGCAGCAAAUAUUGAUUTC	45	GAAAUCAAUAUUUGCUGC	237
M1			ACAGAGAUA
S46-AS238-	CUCUGUGCAGCAAAUAUUGAUUUCT	46	AGAAAUCAAUAUUUGCUG	238
M1			CACAGAGAU
S47-AS239-	UCUGUGCAGCAAAUAUUGAUUUCTA	47	UAGAAAUCAAUAUUUGCU	239
M1			GCACAGAGA
S48-AS240-	CUGUGCAGCAAAUAUUGAUUUCUAC	48	GUAGAAAUCAAUAUUUGC	240
M1			UGCACAGAG
S49-AS241-	UGUGCAGCAAAUAUUGAUUUCUACA	49	UGUAGAAAUCAAUAUUUG	241
M1			CUGCACAGA
S50-AS242-	GUGCAGCAAAUAUUGAUUUCUACAA	50	UUGUAGAAAUCAAUAUUU	242
M1			GCUGCACAG
S51-AS243-	UGCAGCAAAUAUUGAUUUCUACAAC	51	GUUGUAGAAAUCAAUAUU	243
M1			UGCUGCACA
S52-AS244-	GCAGCAAAUAUUGAUUUCUACAACC	52	GGUUGUAGAAAUCAAUAU	244
M1			UUGCUGCAC
S53-AS245-	CAGCAAAUAUUGAUUUCUACAACCA	53	UGGUUGUAGAAAUCAAUA	245
M1			UUUGCUGCA
S54-AS246-	AGCAAAUAUUGAUUUCUACAACCAT	54	AUGGUUGUAGAAAUCAAU	246
M1			AUUUGCUGC
S55-AS247-	GCAAAUAUUGAUUUCUACAACCATC	55	GAUGGUUGUAGAAAUCAA	247
M1			UAUUUGCUG
S56-AS248-	AUAUUGAUUUCUACAACCAUCUUGA	56	UCAAGAUGGUUGUAGAAA	248
M1			UCAAUAUUU
S57-AS249-	UUGAUUUCUACAACCAUCUUGAUAA	57	UUAUCAAGAUGGUUGUAG	249
M1			AAAUCAAUA
S58-AS250-	GAUUUCUACAACCAUCUUGAUAAGT	58	ACUUAUCAAGAUGGUUGU	250
M1			AGAAAUCAA
S59-AS251-	AUUUCUACAACCAUCUUGAUAAGTT	59	AACUUAUCAAGAUGGUUG	251
M1			UAGAAAUCA
S60-AS252-	UUCUACAACCAUCUUGAUAAGUUTA	60	UAAACUUAUCAAGAUGGU	252
M1			UGUAGAAAU
S61-AS253-	CUACAACCAUCUUGAUAAGUUUAAC	61	GUUAAACUUAUCAAGAUG	253
M1			GUUGUAGAA
S62-AS254-	UACAACCAUCUUGAUAAGUUUAACA	62	UGUUAAACUUAUCAAGAU	254
M1			GGUUGUAGA
S63-AS255-	ACAACCAUCUUGAUAAGUUUAACAT	63	AUGUUAAACUUAUCAAGA	255
M1			UGGUUGUAG
S64-AS256-	CAACCAUCUUGAUAAGUUUAACATT	64	AAUGUUAAACUUAUCAAG	256
M1			AUGGUUGUA
S65-AS257-	AACCAUCUUGAUAAGUUUAACAUTG	65	CAAUGUUAAACUUAUCAA	257
M1			GAUGGUUGU
S66-AS258-	ACCAUCUUGAUAAGUUUAACAUUGA	66	UCAAUGUUAAACUUAUCA	258
M1			AGAUGGUUG
S67-AS259-	CCAUCUUGAUAAGUUUAACAUUGAC	67	GUCAAUGUUAAACUUAUC	259
M1			AAGAUGGUU
S68-AS260-	CAUCUUGAUAAGUUUAACAUUGACA	68	UGUCAAUGUUAAACUUAU	260
M1			CAAGAUGGU
S69-AS261-	AUCUUGAUAAGUUUAACAUUGACAA	69	UUGUCAAUGUUAAACUUA	261
M1			UCAAGAUGG
S70-AS262-	GUUCACCACGGUUUCUGAAAUAACA	70	UGUUAUUUCAGAAACCGU	262
M1			GGUGAACAC
S71-AS263-	CACCACGGUUUCUGAAAUAACAGCA	71	UGCUGUUAUUUCAGAAAC	263
M1			CGUGGUGAA
S72-AS264-	CCACGGUUUCUGAAAUAACAGCAAT	72	AUUGCUGUUAUUUCAGAA	264
M1			ACCGUGGUG
S73-AS265-	CACGGUUUCUGAAAUAACAGCAATA	73	UAUUGCUGUUAUUUCAGA	265
M1			AACCGUGGU
S74-AS266-	CGGUUUCUGAAAUAACAGCAAUAGA	74	UCUAUUGCUGUUAUUUCA	266
M1			GAAACCGUG
S75-AS267-	GGUUUCUGAAAUAACAGCAAUAGAA	75	UUCUAUUGCUGUUAUUUC	267
M1			AGAAACCGU
S76-AS268-	GUUUCUGAAAUAACAGCAAUAGAAG	76	CUUCUAUUGCUGUUAUUU	268
M1			CAGAAACCG
S77-AS269-	CUGAAAUAACAGCAAUAGAAGCUGA	77	UCAGCUUCUAUUGCUGUU	269
M1			AUUUCAGAA
S78-AS270-	AAGAGAAAGCCUGAUGUAGUUACTC	78	GAGUAACUACAUCAGGCU	270
M1			UUCUCUUCA
S79-AS271-	AGAGAAAGCCUGAUGUAGUUACUCC	79	GGAGUAACUACAUCAGGC	271
M1			UUUCUCUUC
S80-AS272-	GGCUUGAAUGUUAAGAAAUUUUCAG	80	CUGAAAAUUUCUUAACAU	272
M1			UCAAGCCGU
S81-AS273-	GCUUGAAUGUUAAGAAAUUUUCAGC	81	GCUGAAAAUUUCUUAACA	273
M1			UUCAAGCCG
S82-AS274-	CUUGAAUGUUAAGAAAUUUUCAGCA	82	UGCUGAAAAUUUCUUAAC	274
M1			AUUCAAGCC
S83-AS275-	UUGAAUGUUAAGAAAUUUUCAGCAG	83	CUGCUGAAAAUUUCUUAA	275
M1			CAUUCAAGC
S84-AS276-	UGAAUGUUAAGAAAUUUUCAGCAGT	84	ACUGCUGAAAAUUUCUUA	276
M1			ACAUUCAAG
S85-AS277-	GAAUGUUAAGAAAUUUUCAGCAGTG	85	CACUGCUGAAAAUUUCUU	277
M1			AACAUUCAA
S86-AS278-	AUGUUAAGAAAUUUUCAGCAGUGCA	86	UGCACUGCUGAAAAUUUC	278
M1			UUAACAUUC
S87-AS279-	AGAAAUUUUCAGCAGUGCAUGAGTT	87	AACUCAUGCACUGCUGAAA	279
M1			AUUUCUUA
S88-AS280-	AGCAGUGCAUGAGUUUCAAAAUCTA	88	UAGAUUUUGAAACUCAUG	280
M1			CACUGCUGA
S89-AS281-	AGAUUUUGUUCGAGGUCAUUUCUAT	89	AUAGAAAUGACCUCGAACA	281
M1			AAAUCUUG
S90-AS282-	GUUCGAGGUCAUUUCUAUGGUCATC	90	GAUGACCAUAGAAAUGACC	282
M1			UCGAACAA
S91-AS283-	UUCGAGGUCAUUUCUAUGGUCAUCT	91	AGAUGACCAUAGAAAUGAC	283
M1			CUCGAACA
S92-AS284-	UCGAGGUCAUUUCUAUGGUCAUCTC	92	GAGAUGACCAUAGAAAUG	284
M1			ACCUCGAAC
S93-AS285-	CGAGGUCAUUUCUAUGGUCAUCUCG	93	CGAGAUGACCAUAGAAAUG	285
M1			ACCUCGAA
S94-AS286-	GAGGUCAUUUCUAUGGUCAUCUCGA	94	UCGAGAUGACCAUAGAAA	286
M1			UGACCUCGA
S95-AS287-	AGGUCAUUUCUAUGGUCAUCUCGAC	95	GUCGAGAUGACCAUAGAAA	287
M1			UGACCUCG
S96-AS288-	GGUCAUUUCUAUGGUCAUCUCGACT	96	AGUCGAGAUGACCAUAGAA	288
M1			AUGACCUC
S97-AS289-	GUCAUUUCUAUGGUCAUCUCGACTT	97	AAGUCGAGAUGACCAUAGA	289
M1			AAUGACCU
S98-AS290-	UGAAAAGACUUUGUUCCUUUUCATT	98	AAUGAAAAGGAACAAAGUC	290
M1			UUUUCAAG
S99-AS291-	GAAAAGACUUUGUUCCUUUUCAUTG	99	CAAUGAAAAGGAACAAAGU	291
M1			CUUUUCAA
S100-	AAAGACUUUGUUCCUUUUCAUUGCT	100	AGCAAUGAAAAGGAACAAA	292
AS292-M1			GUCUUUUC
S101-	CUGAGGAUGCAUAAAAGUGACAUCA	101	UGAUGUCACUUUUAUGCA	293
AS293-M1			UCCUCAGCA
S102-	GAGGAUGCAUAAAAGUGACAUCACA	102	UGUGAUGUCACUUUUAUG	294
AS294-M1			CAUCCUCAG
S103-	UUUUUCAUUAUGCCUGCCAAGACAA	103	UUGUCUUGGCAGGCAUAA	295
AS295-M1			UGAAAAACA
S104-	UCAUUAUGCCUGCCAAGACAAAUAA	104	UUAUUUGUCUUGGCAGGC	296
AS296-M1			AUAAUGAAA
S105-	CAUUAUGCCUGCCAAGACAAAUAAT	105	AUUAUUUGUCUUGGCAGG	297
AS297-M1			CAUAAUGAA
S106-	AUUAUGCCUGCCAAGACAAAUAATT	106	AAUUAUUUGUCUUGGCAG	298
AS298-M1			GCAUAAUGA
S107-	AUGCCUGCCAAGACAAAUAAUUUCA	107	UGAAAUUAUUUGUCUUGG	299
AS299-M1			CAGGCAUAA
S108-	AAUUUCAACGUGGAAACCCUGAAAG	108	CUUUCAGGGUUUCCACGU	300
AS300-M1			UGAAAUUAU
S109-	AUUUCAACGUGGAAACCCUGAAAGG	109	CCUUUCAGGGUUUCCACG	301
AS301-M1			UUGAAAUUA
S110-	UUUCAACGUGGAAACCCUGAAAGGA	110	UCCUUUCAGGGUUUCCAC	302
AS302-M1			GUUGAAAUU
S111-	UUCAACGUGGAAACCCUGAAAGGAC	111	GUCCUUUCAGGGUUUCCA	303
AS303-M1			CGUUGAAAU
S112-	UCAACGUGGAAACCCUGAAAGGACA	112	UGUCCUUUCAGGGUUUCC	304
AS304-M1			ACGUUGAAA
S113-	UUGCACAUUCUGUGAAGGAAAAGTT	113	AACUUUUCCUUCACAGAAU	305
AS305-M1			GUGCAACA
S114-	UGCACAUUCUGUGAAGGAAAAGUTT	114	AAACUUUUCCUUCACAGAA	306
AS306-M1			UGUGCAAC
S115-	GCACAUUCUGUGAAGGAAAAGUUTG	115	CAAACUUUUCCUUCACAGA	307
AS307-M1			AUGUGCAA
S116-	AUUCUGUGAAGGAAAAGUUUGGAAA	116	UUUCCAAACUUUUCCUUCA	308
AS308-M1			CAGAAUGU
S117-	GUGAAGGAAAAGUUUGGAAAAAAAC	117	GUUUUUUUCCAAACUUUU	309
AS309-M1			CCUUCACAG
S118-	GAAAAAAACUCUAUGAUGCAUUATT	118	AAUAAUGCAUCAUAGAGU	310
AS310-M1			UUUUUUCCA
S119-	AAAAAAACUCUAUGAUGCAUUAUTA	119	UAAUAAUGCAUCAUAGAG	311
AS311-M1			UUUUUUUCC
S120-	AAAAAACUCUAUGAUGCAUUAUUAA	120	UUAAUAAUGCAUCAUAGA	312
AS312-M1			GUUUUUUUC
S121-	AAAAACUCUAUGAUGCAUUAUUAAG	121	CUUAAUAAUGCAUCAUAG	313
AS313-M1			AGUUUUUUU
S122-	UUAUUAAGAGGAGAAAUUCCUGACC	122	GGUCAGGAAUUUCUCCUC	314
AS314-M1			UUAAUAAUG
S123-	UAUUAAGAGGAGAAAUUCCUGACCT	123	AGGUCAGGAAUUUCUCCU	315
AS315-M1			CUUAAUAAU
S124-	AUUAAGAGGAGAAAUUCCUGACCTG	124	CAGGUCAGGAAUUUCUCC	316
AS316-M1			UCUUAAUAA
S125-	UUAAGAGGAGAAAUUCCUGACCUGA	125	UCAGGUCAGGAAUUUCUC	317
AS317-M1			CUCUUAAUA
S126-	UAAGAGGAGAAAUUCCUGACCUGAA	126	UUCAGGUCAGGAAUUUCU	318
AS318-M1			CCUCUUAAU
S127-	AAGAGGAGAAAUUCCUGACCUGAAC	127	GUUCAGGUCAGGAAUUUC	319
AS319-M1			UCCUCUUAA
S128-	CGAGAUGAUCUAACAAUUAUGAAAA	128	UUUUCAUAAUUGUUAGAU	320
AS320-M1			CAUCUCGAU
S129-	AGAUGAUCUAACAAUUAUGAAAAGA	129	UCUUUUCAUAAUUGUUAG	321
AS321-M1			AUCAUCUCG
S130-	GAUGAUCUAACAAUUAUGAAAAGAG	130	CUCUUUUCAUAAUUGUUA	322
AS322-M1			GAUCAUCUC
S131-	AUGAUCUAACAAUUAUGAAAAGAGC	131	GCUCUUUUCAUAAUUGUU	323
AS323-M1			AGAUCAUCU
S132-	ACAAUUAUGAAAAGAGCCAUCUUTT	132	AAAAGAUGGCUCUUUUCA	324
AS324-M1			UAAUUGUUA
S133-	GAAAAGAGCCAUCUUUUCAACUCAG	133	CUGAGUUGAAAAGAUGGC	325
AS325-M1			UCUUUUCAU
S134-	CCCAUCCUCAGCACCAUUAGACGGA	134	UCCGUCUAAUGGUGCUGA	326
AS326-M1			GGAUGGGGU
S135-	CCAUCCUCAGCACCAUUAGACGGAT	135	AUCCGUCUAAUGGUGCUG	327
AS327-M1			AGGAUGGGG
S136-	CAUCCUCAGCACCAUUAGACGGATT	136	AAUCCGUCUAAUGGUGCU	328
AS328-M1			GAGGAUGGG
S137-	AUCCUCAGCACCAUUAGACGGAUTG	137	CAAUCCGUCUAAUGGUGC	329
AS329-M1			UGAGGAUGG
S138-	UCCUCAGCACCAUUAGACGGAUUGG	138	CCAAUCCGUCUAAUGGUGC	330
AS330-M1			UGAGGAUG
S139-	CCCAUGGACUAUGAAGAGUUUGUTA	139	UAACAAACUCUUCAUAGUC	331
AS331-M1			CAUGGGUA
S140-	CCAUGGACUAUGAAGAGUUUGUUAG	140	CUAACAAACUCUUCAUAGU	332
AS332-M1			CCAUGGGU
S141-	CUUGGAGUAUUUCCAUCAUACUATG	141	CAUAGUAUGAUGGAAAUA	333
AS333-M1			CUCCAAGAU
S142-	UGGAGUAUUUCCAUCAUACUAUGAA	142	UUCAUAGUAUGAUGGAAA	334
AS334-M1			UACUCCAAG
S143-	GGAGUAUUUCCAUCAUACUAUGAAC	143	GUUCAUAGUAUGAUGGAA	335
AS335-M1			AUACUCCAA
S144-	GAGUAUUUCCAUCAUACUAUGAACC	144	GGUUCAUAGUAUGAUGGA	336
AS336-M1			AAUACUCCA
S145-	UAUUUCCAUCAUACUAUGAACCCTG	145	CAGGGUUCAUAGUAUGAU	337
AS337-M1			GGAAAUACU
S146-	AUACUCCAGCUGAAUGCACUGUGAT	146	AUCACAGUGCAUUCAGCUG	338
AS338-M1			GAGUAUAA
S147-	GGCAGAUAUUACCAGCAUGCCAGAC	147	GUCUGGCAUGCUGGUAAU	339
AS339-M1			AUCUGCCUA
S148-	GCAGAUAUUACCAGCAUGCCAGACA	148	UGUCUGGCAUGCUGGUAA	340
AS340-M1			UAUCUGCCU
S149-	CAGAUAUUACCAGCAUGCCAGACAC	149	GUGUCUGGCAUGCUGGUA	341
AS341-M1			AUAUCUGCC
S150-	AGAUAUUACCAGCAUGCCAGACACC	150	GGUGUCUGGCAUGCUGGU	342
AS342-M1			AAUAUCUGC
S151-	GAUAUUACCAGCAUGCCAGACACCT	151	AGGUGUCUGGCAUGCUGG	343
AS343-M1			UAAUAUCUG
S152-	AUAUUACCAGCAUGCCAGACACCTG	152	CAGGUGUCUGGCAUGCUG	344
AS344-M1			GUAAUAUCU
S153-	UAUUACCAGCAUGCCAGACACCUGA	153	UCAGGUGUCUGGCAUGCU	345
AS345-M1			GGUAAUAUC
S154-	AUUACCAGCAUGCCAGACACCUGAC	154	GUCAGGUGUCUGGCAUGC	346
AS346-M1			UGGUAAUAU
S155-	UUACCAGCAUGCCAGACACCUGACA	155	UGUCAGGUGUCUGGCAUG	347
AS347-M1			CUGGUAAUA
S156-	UACCAGCAUGCCAGACACCUGACAT	156	AUGUCAGGUGUCUGGCAU	348
AS348-M1			GCUGGUAAU
S157-	ACCAGCAUGCCAGACACCUGACATT	157	AAUGUCAGGUGUCUGGCA	349
AS349-M1			UGCUGGUAA
S158-	CCAGCAUGCCAGACACCUGACAUTA	158	UAAUGUCAGGUGUCUGGC	350
AS350-M1			AUGCUGGUA
S159-	CAGCAUGCCAGACACCUGACAUUAA	159	UUAAUGUCAGGUGUCUGG	351
AS351-M1			CAUGCUGGU
S160-	AGCAUGCCAGACACCUGACAUUAAG	160	CUUAAUGUCAGGUGUCUG	352
AS352-M1			GCAUGCUGG
S161-	GCAUGCCAGACACCUGACAUUAAGC	161	GCUUAAUGUCAGGUGUCU	353
AS353-M1			GGCAUGCUG
S162-	UGCCAGACACCUGACAUUAAGCAGA	162	UCUGCUUAAUGUCAGGUG	354
AS354-M1			UCUGGCAUG
S163-	CCUGACAUUAAGCAGAGCUUUUCCA	163	UGGAAAAGCUCUGCUUAA	355
AS355-M1			UGUCAGGUG
S164-	AAGCAGAGCUUUUCCAGAUAAAUTC	164	GAAUUUAUCUGGAAAAGC	356
AS356-M1			UCUGCUUAA
S165-	GCAGAGCUUUUCCAGAUAAAUUCCA	165	UGGAAUUUAUCUGGAAAA	357
AS357-M1			GCUCUGCUU
S166-	CCAGAUAAAUUCCAUGUGGAACUAA	166	UUAGUUCCACAUGGAAUU	358
AS358-M1			UAUCUGGAA
S167-	CAGAUAAAUUCCAUGUGGAACUAAC	167	GUUAGUUCCACAUGGAAU	359
AS359-M1			UUAUCUGGA
S168-	UCCUCAGUACCACCUUCUCCUUCAG	168	CUGAAGGAGAAGGUGGUA	360
AS360-M1			CUGAGGAAG
S169-	CCUCAGUACCACCUUCUCCUUCAGG	169	CCUGAAGGAGAAGGUGGU	361
AS361-M1			ACUGAGGAA
S170-	GAAAGGGAUCGGUUAAAUAUCAAGT	170	ACUUGAUAUUUAACCGAU	362
AS362-M1			CCCUUUCAG
S171-	AGGGAUCGGUUAAAUAUCAAGUCAC	171	GUGACUUGAUAUUUAACC	363
AS363-M1			GAUCCCUUU
S172-	AUCGGUUAAAUAUCAAGUCACCATT	172	AAUGGUGACUUGAUAUUU	364
AS364-M1			AACCGAUCC
S173-	GGUUAAAUAUCAAGUCACCAUUUTC	173	GAAAAUGGUGACUUGAUA	365
AS365-M1			UUUAACCGA
S174-	GUUAAAUAUCAAGUCACCAUUUUCA	174	UGAAAAUGGUGACUUGAU	366
AS366-M1			AUUUAACCG
S175-	AAAUAUCAAGUCACCAUUUUCACTG	175	CAGUGAAAAUGGUGACUU	367
AS367-M1			GAUAUUUAA
S176-	AAGAAAAAGCUGCAUGGUGAAUATA	176	UAUAUUCACCAUGCAGCU	368
AS368-M1			UUUUCUUCC
S177-	AGAAAAAGCUGCAUGGUGAAUAUAA	177	UUAUAUUCACCAUGCAGC	369
AS369-M1			UUUUUCUUC
S178-	AAAAAGCUGCAUGGUGAAUAUAAGA	178	UCUUAUAUUCACCAUGCA	370
AS370-M1			GCUUUUUCU
S179-	AAAAGCUGCAUGGUGAAUAUAAGAA	179	UUCUUAUAUUCACCAUGC	371
AS371-M1			AGCUUUUUC
S180-	AAGCUGCAUGGUGAAUAUAAGAACT	180	AGUUCUUAUAUUCACCAU	372
AS372-M1			GCAGCUUUU
S181-	GCUGCAUGGUGAAUAUAAGAACUGA	181	UCAGUUCUUAUAUUCACC	373
AS373-M1			AUGCAGCUU
S182-	CUGCAUGGUGAAUAUAAGAACUGAA	182	UUCAGUUCUUAUAUUCAC	374
AS374-M1			CAUGCAGCU
S183-	GCAUGGUGAAUAUAAGAACUGAATT	183	AAUUCAGUUCUUAUAUUC	375
AS375-M1			ACCAUGCAG
S184-	CAUGGUGAAUAUAAGAACUGAAUTC	184	GAAUUCAGUUCUUAUAUU	376
AS376-M1			CACCAUGCA
S185-	AUGGUGAAUAUAAGAACUGAAUUCT	185	AGAAUUCAGUUCUUAUAU	377
AS377-M1			UCACCAUGC
S186-	UGGUGAAUAUAAGAACUGAAUUCTA	186	UAGAAUUCAGUUCUUAUA	378
AS378-M1			UUCACCAUG
S187-	GGUGAAUAUAAGAACUGAAUUCUAC	187	GUAGAAUUCAGUUCUUAU	379
AS379-M1			AUUCACCAU
S188-	GUGAAUAUAAGAACUGAAUUCUACA	188	UGUAGAAUUCAGUUCUUA	380
AS380-M1			UAUUCACCA
S189-	AAUAUAAGAACUGAAUUCUACAUGT	189	ACAUGUAGAAUUCAGUUC	381
AS381-M1			UUAUAUUCA
S190-	AUAUAAGAACUGAAUUCUACAUGTG	190	CACAUGUAGAAUUCAGUU	382
AS382-M1			CUUAUAUUC
S191-	AUAAGAACUGAAUUCUACAUGUGCT	191	AGCACAUGUAGAAUUCAG	383
AS383-M1			UUCUUAUAU
S192-	AAGAACUGAAUUCUACAUGUGCUGC	192	GCAGCACAUGUAGAAUUCA	384
AS384-M1			GUUCUUAU
S385-	AGGUGCAUUUUGGAAGAUGGGCAGC	385	CCAUCUUCCAAAAUGCACC	417
AS417-M2	CGAAAGGCUGC		UGG
S385-	AGGUGCAUUUUGGAAGAUGGGCAGC	385	CCAUCUUCCAAAAUGCACC	417
AS417-M3	CGAAAGGCUGC		UGG
S385-	AGGUGCAUUUUGGAAGAUGGGCAGC	385	CCAUCUUCCAAAAUGCACC	417
AS417-M4	CGAAAGGCUGC		UGG
S385-	AGGUGCAUUUUGGAAGAUGGGCAGC	385	CCAUCUUCCAAAAUGCACC	417
AS417-M5	CGAAAGGCUGC		UGG
S385-	AGGUGCAUUUUGGAAGAUGGGCAGC	385	CCAUCUUCCAAAAUGCACC	417
AS417-M6	CGAAAGGCUGC		UGG
S385-	AGGUGCAUUUUGGAAGAUGGGCAGC	385	CCAUCUUCCAAAAUGCACC	417
AS417-M7	CGAAAGGCUGC		UGG
S385-	AGGUGCAUUUUGGAAGAUGGGCAGC	385	CCAUCUUCCAAAAUGCACC	417
AS417-M8	CGAAAGGCUGC		UGG
S385-	AGGUGCAUUUUGGAAGAUGGGCAGC	385	CCAUCUUCCAAAAUGCACC	417
AS417-M9	CGAAAGGCUGC		UGG
S385-	AGGUGCAUUUUGGAAGAUGGGCAGC	385	CCAUCUUCCAAAAUGCACC	417
AS417-M10	CGAAAGGCUGC		UGG
S385-	AGGUGCAUUUUGGAAGAUGGGCAGC	385	CCAUCUUCCAAAAUGCACC	417
AS417-M11	CGAAAGGCUGC		UGG
S386-	AGGUGCAUUUUGGAAGAUGGCAGCC	386	CAUCUUCCAAAAUGCACCU	418
AS418-M12	GAAAGGCUGC		GG
S386-	AGGUGCAUUUUGGAAGAUGGCAGCC	386	CAUCUUCCAAAAUGCACCU	418
AS418-M13	GAAAGGCUGC		GG
S387-	GGUGCAUUUUGGAAGAUGGCGCAGCC	387	GCCAUCUUCCAAAAUGCAC	419
AS419-M2	GAAAGGCUGC		CUG
S387-	GGUGCAUUUUGGAAGAUGGCGCAGCC	387	GCCAUCUUCCAAAAUGCAC	419
AS419-M3	GAAAGGCUGC		CUG
S387-	GGUGCAUUUUGGAAGAUGGCGCAGCC	387	GCCAUCUUCCAAAAUGCAC	419
AS419-M4	GAAAGGCUGC		CUG
S387-	GGUGCAUUUUGGAAGAUGGCGCAGCC	387	GCCAUCUUCCAAAAUGCAC	419
AS419-M5	GAAAGGCUGC		CUG
S387-	GGUGCAUUUUGGAAGAUGGCGCAGCC	387	GCCAUCUUCCAAAAUGCAC	419
AS419-M6	GAAAGGCUGC		CUG
S387-	GGUGCAUUUUGGAAGAUGGCGCAGCC	387	GCCAUCUUCCAAAAUGCAC	419
AS419-M7	GAAAGGCUGC		CUG
S387-	GGUGCAUUUUGGAAGAUGGCGCAGCC	387	GCCAUCUUCCAAAAUGCAC	419
AS419-M8	GAAAGGCUGC		CUG
S387-	GGUGCAUUUUGGAAGAUGGCGCAGCC	387	GCCAUCUUCCAAAAUGCAC	419
AS419-M9	GAAAGGCUGC		CUG
S387-	GGUGCAUUUUGGAAGAUGGCGCAGCC	387	GCCAUCUUCCAAAAUGCAC	419
AS419-M10	GAAAGGCUGC		CUG
S387-	GGUGCAUUUUGGAAGAUGGCGCAGCC	387	GCCAUCUUCCAAAAUGCAC	419
AS419-M11	GAAAGGCUGC		CUG
S388-	GGUGCAUUUUGGAAGAUGGGCAGCC	388	CCAUCUUCCAAAAUGCACC	420
AS420-M12	GAAAGGCUGC		UG
S388-	GGUGCAUUUUGGAAGAUGGGCAGCC	388	CCAUCUUCCAAAAUGCACC	420
AS420-M13	GAAAGGCUGC		UG
S389-	AAGGAAGUCCUUAUGUGGUAGCAGCC	389	UACCACAUAAGGACUUCCU	421
AS421-M2	GAAAGGCUGC		UCU
S389-	AAGGAAGUCCUUAUGUGGUAGCAGCC	389	UACCACAUAAGGACUUCCU	421
AS421-M3	GAAAGGCUGC		UCU
S389-	AAGGAAGUCCUUAUGUGGUAGCAGCC	389	UACCACAUAAGGACUUCCU	421
AS421-M4	GAAAGGCUGC		UCU
S389-	AAGGAAGUCCUUAUGUGGUAGCAGCC	389	UACCACAUAAGGACUUCCU	421
AS421-M5	GAAAGGCUGC		UCU
S389-	AAGGAAGUCCUUAUGUGGUAGCAGCC	389	UACCACAUAAGGACUUCCT	422
AS422-M6	GAAAGGCUGC		UCU
S389-	AAGGAAGUCCUUAUGUGGUAGCAGCC	389	UACCACAUAAGGACUUCCU	421
AS421-M7	GAAAGGCUGC		UCU
S389-	AAGGAAGUCCUUAUGUGGUAGCAGCC	389	UACCACAUAAGGACUUCCU	421
AS421-M8	GAAAGGCUGC		UCU
S389-	AAGGAAGUCCUUAUGUGGUAGCAGCC	389	UACCACAUAAGGACUUCCU	421
AS421-M9	GAAAGGCUGC		UCU
S389-	AAGGAAGUCCUUAUGUGGUAGCAGCC	389	UACCACAUAAGGACUUCCU	421
AS421-M10	GAAAGGCUGC		UCU
S389-	AAGGAAGUCCUUAUGUGGUAGCAGCC	389	UACCACAUAAGGACUUCCU	421
AS421-M11	GAAAGGCUGC		UCU
S390-	AAGGAAGUCCUUAUGUGGUGCAGCCG	390	ACCACAUAAGGACUUCCTU	423
AS423-M12	AAAGGCUGC		CU
S390-	AAGGAAGUCCUUAUGUGGUGCAGCCG	390	ACCACAUAAGGACUUCCUU	424
AS424-M13	AAAGGCUGC		CU
S391-	AUGAUAUGCUGAUAUUUGGAGCAGC	391	UCCAAAUAUCAGCAUAUCA	425
AS425-M2	CGAAAGGCUGC		UUG
S391-	AUGAUAUGCUGAUAUUUGGAGCAGC	391	UCCAAAUAUCAGCAUAUCA	425
AS425-M3	CGAAAGGCUGC		UUG
S391-	AUGAUAUGCUGAUAUUUGGAGCAGC	391	UCCAAAUAUCAGCAUAUCA	425
AS425-M4	CGAAAGGCUGC		UUG
S391-	AUGAUAUGCUGAUAUUUGGAGCAGC	391	UCCAAAUAUCAGCAUAUCA	425
AS425-M5	CGAAAGGCUGC		UUG
S391-	AUGAUAUGCUGAUAUUUGGAGCAGC	391	UCCAAAUAUCAGCAUATCA	426
AS426-M6	CGAAAGGCUGC		UUG
S391-	AUGAUAUGCUGAUAUUUGGAGCAGC	391	UCCAAAUAUCAGCAUAUCA	425
AS425-M7	CGAAAGGCUGC		UUG
S391-	AUGAUAUGCUGAUAUUUGGAGCAGC	391	UCCAAAUAUCAGCAUAUCA	425
AS425-M8	CGAAAGGCUGC		UUG
S391-	AUGAUAUGCUGAUAUUUGGAGCAGC	391	UCCAAAUAUCAGCAUAUCA	425
AS425-M9	CGAAAGGCUGC		UUG
S391-	AUGAUAUGCUGAUAUUUGGAGCAGC	391	UCCAAAUAUCAGCAUAUCA	425
AS425-M10	CGAAAGGCUGC		UUG
S391-	AUGAUAUGCUGAUAUUUGGAGCAGC	391	UCCAAAUAUCAGCAUAUCA	425
AS425-M11	CGAAAGGCUGC		UUG
S392-	AUGAUAUGCUGAUAUUUGGGCAGCC	392	CCAAAUAUCAGCAUATCAU	427
AS427-M12	GAAAGGCUGC		UG
S392-	AUGAUAUGCUGAUAUUUGGGCAGCC	392	CCAAAUAUCAGCAUAUCAU	428
AS428-M13	GAAAGGCUGC		UG
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAAUAUUUG	429
AS429-M2	GAAAGGCUGC		CUGC
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAAUAUUUG	429
AS429-M3	GAAAGGCUGC		CUGC
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAAUAUUUG	429
AS429-M4	GAAAGGCUGC		CUGC
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAAUAUUUG	429
AS429-M5	GAAAGGCUGC		CUGC
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAATAUUTGC	430
AS430-M6	GAAAGGCUGC		UGC
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAAUAUUUG	429
AS429-M7	GAAAGGCUGC		CUGC
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAAUAUUUG	429
AS429-M8	GAAAGGCUGC		CUGC
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAAUAUUUG	429
AS429-M9	GAAAGGCUGC		CUGC
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAAUAUUUG	429
AS429-M10	GAAAGGCUGC		CUGC
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAAUAUUUG	429
AS429-M11	GAAAGGCUGC		CUGC
S394-	AGCAAAUAUUGAUUUCUACGCAGCCG	394	GTAGAAAUCAATAUUTGCU	431
AS431-M12	AAAGGCUGC		GC
S394-	AGCAAAUAUUGAUUUCUACGCAGCCG	394	GUAGAAAUCAAUAUUUGC	432
AS432-M13	AAAGGCUGC		UGC
S395-	CCACGGUUUCUGAAAUAACAGCAGCC	395	UGUUAUUUCAGAAACCGU	433
AS433-M2	GAAAGGCUGC		GGUG
S395-	CCACGGUUUCUGAAAUAACAGCAGCC	395	UGUUAUUUCAGAAACCGU	433
AS433-M3	GAAAGGCUGC		GGUG
S395-	CCACGGUUUCUGAAAUAACAGCAGCC	395	UGUUAUUUCAGAAACCGU	433
AS433-M4	GAAAGGCUGC		GGUG
S395-	CCACGGUUUCUGAAAUAACAGCAGCC	395	UGUUAUUUCAGAAACCGU	433
AS433-M5	GAAAGGCUGC		GGUG
S395-	CCACGGUUUCUGAAAUAACAGCAGCC	395	UGUUAUUUCAGAAACCGU	433
AS433-M6	GAAAGGCUGC		GGUG
S395-	CCACGGUUUCUGAAAUAACAGCAGCC	395	UGUUAUUUCAGAAACCGU	433
AS433-M7	GAAAGGCUGC		GGUG
S395-	CCACGGUUUCUGAAAUAACAGCAGCC	395	UGUUAUUUCAGAAACCGU	433
AS433-M8	GAAAGGCUGC		GGUG
S395-	CCACGGUUUCUGAAAUAACAGCAGCC	395	UGUUAUUUCAGAAACCGU	433
AS433-M9	GAAAGGCUGC		GGUG
S395-	CCACGGUUUCUGAAAUAACAGCAGCC	395	UGUUAUUUCAGAAACCGU	433
AS433-M10	GAAAGGCUGC		GGUG
S395-	CCACGGUUUCUGAAAUAACAGCAGCC	395	UGUUAUUUCAGAAACCGU	433
AS433-M11	GAAAGGCUGC		GGUG
S396-	CCACGGUUUCUGAAAUAACGCAGCCG	396	GTUAUUUCAGAAACCGUG	434
AS434-M12	AAAGGCUGC		GUG
S396-	CCACGGUUUCUGAAAUAACGCAGCCG	396	GUUAUUUCAGAAACCGUG	435
AS435-M13	AAAGGCUGC		GUG
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	436
AS436-M2	GAAAGGCUGC		GCC
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	436
AS436-M3	GAAAGGCUGC		GCC
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	436
AS436-M4	GAAAGGCUGC		GCC
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	436
AS436-M5	GAAAGGCUGC		GCC
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	436
AS436-M6	GAAAGGCUGC		GCC
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	436
AS436-M7	GAAAGGCUGC		GCC
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	436
AS436-M8	GAAAGGCUGC		GCC
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	436
AS436-M9	GAAAGGCUGC		GCC
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	436
AS436-M10	GAAAGGCUGC		GCC
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	436
AS436-M11	GAAAGGCUGC		GCC
S398-	CUUGAAUGUUAAGAAAUUUGCAGCCG	398	AAAUUUCUUAACAUUCAA	437
AS437-M12	AAAGGCUGC		GCC
S398-	CUUGAAUGUUAAGAAAUUUGCAGCCG	398	AAAUUUCUUAACAUUCAA	437
AS437-M13	AAAGGCUGC		GCC
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	438
AS438-M2	GAAAGGCUGC		CAAG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	438
AS438-M3	GAAAGGCUGC		CAAG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	438
AS438-M4	GAAAGGCUGC		CAAG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	438
AS438-M5	GAAAGGCUGC		CAAG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACATUC	439
AS439-M6	GAAAGGCUGC		AAG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	438
AS438-M7	GAAAGGCUGC		CAAG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	438
AS438-M8	GAAAGGCUGC		CAAG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	438
AS438-M9	GAAAGGCUGC		CAAG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	438
AS438-M10	GAAAGGCUGC		CAAG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	438
AS438-M11	GAAAGGCUGC		CAAG
S400-	UGAAUGUUAAGAAAUUUUCGCAGCCG	400	GAAAAUUUCUUAACATUCA	440
AS440-M12	AAAGGCUGC		AG
S400-	UGAAUGUUAAGAAAUUUUCGCAGCCG	400	GAAAAUUUCUUAACAUUC	441
AS441-M13	AAAGGCUGC		AAG
S401-	CGAGGUCAUUUCUAUGGUCAGCAGCC	401	UGACCAUAGAAAUGACCUC	442
AS442-M2	GAAAGGCUGC		GAA
S401-	CGAGGUCAUUUCUAUGGUCAGCAGCC	401	UGACCAUAGAAAUGACCUC	442
AS442-M3	GAAAGGCUGC		GAA
S401-	CGAGGUCAUUUCUAUGGUCAGCAGCC	401	UGACCAUAGAAAUGACCUC	442
AS442-M4	GAAAGGCUGC		GAA
S401-	CGAGGUCAUUUCUAUGGUCAGCAGCC	401	UGACCAUAGAAAUGACCUC	442
AS442-M5	GAAAGGCUGC		GAA
S401-	CGAGGUCAUUUCUAUGGUCAGCAGCC	401	UGACCAUAGAAATGACCUC	443
AS443-M6	GAAAGGCUGC		GAA
S401-	CGAGGUCAUUUCUAUGGUCAGCAGCC	401	UGACCAUAGAAAUGACCUC	442
AS442-M7	GAAAGGCUGC		GAA
S401-	CGAGGUCAUUUCUAUGGUCAGCAGCC	401	UGACCAUAGAAAUGACCUC	442
AS442-M8	GAAAGGCUGC		GAA
S401-	CGAGGUCAUUUCUAUGGUCAGCAGCC	401	UGACCAUAGAAAUGACCUC	442
AS442-M9	GAAAGGCUGC		GAA
S401-	CGAGGUCAUUUCUAUGGUCAGCAGCC	401	UGACCAUAGAAAUGACCUC	442
AS442-M10	GAAAGGCUGC		GAA
S401-	CGAGGUCAUUUCUAUGGUCAGCAGCC	401	UGACCAUAGAAAUGACCUC	442
AS442-M11	GAAAGGCUGC		GAA
S402-	CGAGGUCAUUUCUAUGGUCGCAGCCG	402	GACCAUAGAAATGACCUCG	444
AS444-M12	AAAGGCUGC		AA
S402-	CGAGGUCAUUUCUAUGGUCGCAGCCG	402	GACCAUAGAAAUGACCUCG	445
AS445-M13	AAAGGCUGC		AA
S403-	AUUCUGUGAAGGAAAAGUUUGCAGCC	403	AAACUUUUCCUUCACAGAA	446
AS446-M2	GAAAGGCUGC		UGU
S403-	AUUCUGUGAAGGAAAAGUUUGCAGCC	403	AAACUUUUCCUUCACAGAA	446
AS446-M3	GAAAGGCUGC		UGU
S403-	AUUCUGUGAAGGAAAAGUUUGCAGCC	403	AAACUUUUCCUUCACAGAA	446
AS446-M4	GAAAGGCUGC		UGU
S403-	AUUCUGUGAAGGAAAAGUUUGCAGCC	403	AAACUUUUCCUUCACAGAA	446
AS446-M5	GAAAGGCUGC		UGU
S403-	AUUCUGUGAAGGAAAAGUUUGCAGCC	403	AAACUUUUCCUUCACAGAA	446
AS446-M6	GAAAGGCUGC		UGU
S403-	AUUCUGUGAAGGAAAAGUUUGCAGCC	403	AAACUUUUCCUUCACAGAA	446
AS446-M7	GAAAGGCUGC		UGU
S403-	AUUCUGUGAAGGAAAAGUUUGCAGCC	403	AAACUUUUCCUUCACAGAA	446
AS446-M8	GAAAGGCUGC		UGU
S403-	AUUCUGUGAAGGAAAAGUUUGCAGCC	403	AAACUUUUCCUUCACAGAA	446
AS446-M9	GAAAGGCUGC		UGU
S403-	AUUCUGUGAAGGAAAAGUUUGCAGCC	403	AAACUUUUCCUUCACAGAA	446
AS446-M10	GAAAGGCUGC		UGU
S403-	AUUCUGUGAAGGAAAAGUUUGCAGCC	403	AAACUUUUCCUUCACAGAA	446
AS446-M11	GAAAGGCUGC		UGU
S404-	AUUCUGUGAAGGAAAAGUUGCAGCCG	404	AACUUUUCCUUCACAGAAU	447
AS447-M12	AAAGGCUGC		GU
S404-	AUUCUGUGAAGGAAAAGUUGCAGCCG	404	AACUUUUCCUUCACAGAAU	447
AS447-M13	AAAGGCUGC		GU
S405-	CUUGGAGUAUUUCCAUCAUAGCAGCC	405	UAUGAUGGAAAUACUCCA	448
AS448-M2	GAAAGGCUGC		AGAU
S405-	CUUGGAGUAUUUCCAUCAUAGCAGCC	405	UAUGAUGGAAAUACUCCA	448
AS448-M3	GAAAGGCUGC		AGAU
S405-	CUUGGAGUAUUUCCAUCAUAGCAGCC	405	UAUGAUGGAAAUACUCCA	448
AS448-M4	GAAAGGCUGC		AGAU
S405-	CUUGGAGUAUUUCCAUCAUAGCAGCC	405	UAUGAUGGAAAUACUCCA	448
AS448-M5	GAAAGGCUGC		AGAU
S405-	CUUGGAGUAUUUCCAUCAUAGCAGCC	405	UAUGAUGGAAAUACUCCA	448
AS448-M6	GAAAGGCUGC		AGAU
S405-	CUUGGAGUAUUUCCAUCAUAGCAGCC	405	UAUGAUGGAAAUACUCCA	448
AS448-M7	GAAAGGCUGC		AGAU
S405-	CUUGGAGUAUUUCCAUCAUAGCAGCC	405	UAUGAUGGAAAUACUCCA	448
AS448-M8	GAAAGGCUGC		AGAU
S405-	CUUGGAGUAUUUCCAUCAUAGCAGCC	405	UAUGAUGGAAAUACUCCA	448
AS448-M9	GAAAGGCUGC		AGAU
S405-	CUUGGAGUAUUUCCAUCAUAGCAGCC	405	UAUGAUGGAAAUACUCCA	448
AS448-M10	GAAAGGCUGC		AGAU
S405-	CUUGGAGUAUUUCCAUCAUAGCAGCC	405	UAUGAUGGAAAUACUCCA	448
AS448-M11	GAAAGGCUGC		AGAU
S406-	CUUGGAGUAUUUCCAUCAUGCAGCCG	406	ATGAUGGAAAUACUCCAAG	449
AS449-M12	AAAGGCUGC		AU
S406-	CUUGGAGUAUUUCCAUCAUGCAGCCG	406	AUGAUGGAAAUACUCCAA	450
AS450-M13	AAAGGCUGC		GAU
S407-	UGGAGUAUUUCCAUCAUACUGCAGCC	407	AGUAUGAUGGAAAUACUC	451
AS451-M2	GAAAGGCUGC		CAAG
S407-	UGGAGUAUUUCCAUCAUACUGCAGCC	407	AGUAUGAUGGAAAUACUC	451
AS451-M3	GAAAGGCUGC		CAAG
S407-	UGGAGUAUUUCCAUCAUACUGCAGCC	407	AGUAUGAUGGAAAUACUC	451
AS451-M4	GAAAGGCUGC		CAAG
S407-	UGGAGUAUUUCCAUCAUACUGCAGCC	407	AGUAUGAUGGAAAUACUC	451
AS451-M5	GAAAGGCUGC		CAAG
S407-	UGGAGUAUUUCCAUCAUACUGCAGCC	407	AGUAUGAUGGAAAUACTCC	452
AS452-M6	GAAAGGCUGC		AAG
S407-	UGGAGUAUUUCCAUCAUACUGCAGCC	407	AGUAUGAUGGAAAUACUC	451
AS451-M7	GAAAGGCUGC		CAAG
S407-	UGGAGUAUUUCCAUCAUACUGCAGCC	407	AGUAUGAUGGAAAUACUC	451
AS451-M8	GAAAGGCUGC		CAAG
S407-	UGGAGUAUUUCCAUCAUACUGCAGCC	407	AGUAUGAUGGAAAUACUC	451
AS451-M9	GAAAGGCUGC		CAAG
S407-	UGGAGUAUUUCCAUCAUACUGCAGCC	407	AGUAUGAUGGAAAUACUC	451
AS451-M10	GAAAGGCUGC		CAAG
S407-	UGGAGUAUUUCCAUCAUACUGCAGCC	407	AGUAUGAUGGAAAUACUC	451
AS451-M11	GAAAGGCUGC		CAAG
S408-	UGGAGUAUUUCCAUCAUACGCAGCCG	408	GTAUGAUGGAAAUACTCCA	453
AS453-M12	AAAGGCUGC		AG
S408-	UGGAGUAUUUCCAUCAUACGCAGCCG	408	GUAUGAUGGAAAUACUCC	454
AS454-M13	AAAGGCUGC		AAG
S409-	GAGUAUUUCCAUCAUACUAUGCAGCC	409	AUAGUAUGAUGGAAAUAC	455
AS455-M2	GAAAGGCUGC		UCCA
S409-	GAGUAUUUCCAUCAUACUAUGCAGCC	409	AUAGUAUGAUGGAAAUAC	455
AS455-M3	GAAAGGCUGC		UCCA
S409-	GAGUAUUUCCAUCAUACUAUGCAGCC	409	AUAGUAUGAUGGAAAUAC	455
AS455-M4	GAAAGGCUGC		UCCA
S409-	GAGUAUUUCCAUCAUACUAUGCAGCC	409	AUAGUAUGAUGGAAAUAC	455
AS455-M5	GAAAGGCUGC		UCCA
S409-	GAGUAUUUCCAUCAUACUAUGCAGCC	409	ATAGUAUGAUGGAAAUACT	456
AS456-M6	GAAAGGCUGC		CCA
S409-	GAGUAUUUCCAUCAUACUAUGCAGCC	409	AUAGUAUGAUGGAAAUAC	455
AS455-M7	GAAAGGCUGC		UCCA
S409-	GAGUAUUUCCAUCAUACUAUGCAGCC	409	AUAGUAUGAUGGAAAUAC	455
AS455-M8	GAAAGGCUGC		UCCA
S409-	GAGUAUUUCCAUCAUACUAUGCAGCC	409	AUAGUAUGAUGGAAAUAC	455
AS455-M9	GAAAGGCUGC		UCCA
S409-	GAGUAUUUCCAUCAUACUAUGCAGCC	409	AUAGUAUGAUGGAAAUAC	455
AS455-M10	GAAAGGCUGC		UCCA
S409-	GAGUAUUUCCAUCAUACUAUGCAGCC	409	AUAGUAUGAUGGAAAUAC	455
AS455-M11	GAAAGGCUGC		UCCA
S410-	GAGUAUUUCCAUCAUACUAGCAGCCG	410	UAGUAUGAUGGAAAUACT	457
AS457-M12	AAAGGCUGC		CCA
S410-	GAGUAUUUCCAUCAUACUAGCAGCCG	410	UAGUAUGAUGGAAAUACU	458
AS458-M13	AAAGGCUGC		CCA
S411-	CCAGCAUGCCAGACACCUGAGCAGCC	411	UCAGGUGUCUGGCAUGCU	459
AS459-M2	GAAAGGCUGC		GGUA
S411-	CCAGCAUGCCAGACACCUGAGCAGCC	411	UCAGGUGUCUGGCAUGCU	459
AS459-M3	GAAAGGCUGC		GGUA
S411-	CCAGCAUGCCAGACACCUGAGCAGCC	411	UCAGGUGUCUGGCAUGCU	459
AS459-M4	GAAAGGCUGC		GGUA
S411-	CCAGCAUGCCAGACACCUGAGCAGCC	411	UCAGGUGUCUGGCAUGCU	459
AS459-M5	GAAAGGCUGC		GGUA
S411-	CCAGCAUGCCAGACACCUGAGCAGCC	411	UCAGGUGUCUGGCAUGCU	459
AS459-M6	GAAAGGCUGC		GGUA
S411-	CCAGCAUGCCAGACACCUGAGCAGCC	411	UCAGGUGUCUGGCAUGCU	459
AS459-M7	GAAAGGCUGC		GGUA
S411-	CCAGCAUGCCAGACACCUGAGCAGCC	411	UCAGGUGUCUGGCAUGCU	459
AS459-M8	GAAAGGCUGC		GGUA
S411-	CCAGCAUGCCAGACACCUGAGCAGCC	411	UCAGGUGUCUGGCAUGCU	459
AS459-M9	GAAAGGCUGC		GGUA
S411-	CCAGCAUGCCAGACACCUGAGCAGCC	411	UCAGGUGUCUGGCAUGCU	459
AS459-M10	GAAAGGCUGC		GGUA
S411-	CCAGCAUGCCAGACACCUGAGCAGCC	411	UCAGGUGUCUGGCAUGCU	459
AS459-M11	GAAAGGCUGC		GGUA
S412-	CCAGCAUGCCAGACACCUGGCAGCCG	412	CAGGUGUCUGGCAUGCUG	460
AS460-M12	AAAGGCUGC		GUA
S412-	CCAGCAUGCCAGACACCUGGCAGCCG	412	CAGGUGUCUGGCAUGCUG	460
AS460-M13A	AAGGCUGC		GUA
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	461
AS461-M2	GAAAGGCUGC		CUUC
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	461
AS461-M3	GAAAGGCUGC		CUUC
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	461
AS461-M4	GAAAGGCUGC		CUUC
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	461
AS461-M5	GAAAGGCUGC		CUUC
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UTCACCAUGCAGCUUUTUC	462
AS462-M6	GAAAGGCUGC		UUC
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	461
AS461-M7	GAAAGGCUGC		CUUC
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	461
AS461-M8	GAAAGGCUGC		CUUC
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	461
AS461-M9	GAAAGGCUGC		CUUC
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	461
AS461-M10	GAAAGGCUGC		CUUC
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	461
AS461-M11	GAAAGGCUGC		CUUC
S414-	AGAAAAAGCUGCAUGGUGAGCAGCCG	414	UCACCAUGCAGCUUUTUCU	463
AS463-M12	AAAGGCUGC		UC
S414-	AGAAAAAGCUGCAUGGUGAGCAGCCG	414	UCACCAUGCAGCUUUUUC	464
AS464-M13	AAAGGCUGC		UUC
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	465
AS465-M2	GAAAGGCUGC		CAUG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	465
AS465-M3	GAAAGGCUGC		CAUG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	465
AS465-M4	GAAAGGCUGC		CAUG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	465
AS465-M5	GAAAGGCUGC		CAUG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UTCAGUUCUUAUAUUCACC	598
AS598-M6	GAAAGGCUGC		AUG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	465
AS465-M7	GAAAGGCUGC		CAUG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	465
AS465-M8	GAAAGGCUGC		CAUG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	465
AS465-M9	GAAAGGCUGC		CAUG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	465
AS465-M10	GAAAGGCUGC		CAUG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	465
AS465-M11	GAAAGGCUGC		CAUG
S416-	UGGUGAAUAUAAGAACUGAGCAGCCG	416	UCAGUUCUUAUAUUCACC	466
AS466-M12	AAAGGCUGC		AUG
S416-	UGGUGAAUAUAAGAACUGAGCAGCCG	416	UCAGUUCUUAUAUUCACC	466
AS466-M13	AAAGGCUGC		AUG
S467-	GGAGGCAUCUAUACUGUGAUUCAGA	467	UCUGAAUCACAGUAUAGA	518
AS518-M1			UGCCUCCAA
S468-	GGCAUCUAUACUGUGAUUCAGACAA	468	UUGUCUGAAUCACAGUAU	519
AS519-M1			AGAUGCCUC
S469-	GCAUCUAUACUGUGAUUCAGACAAA	469	UUUGUCUGAAUCACAGUA	520
AS520-M1			UAGAUGCCU
S470-	GUCCAUAUUUUGAGCAUAAUAUGAA	470	UUCAUAUUAUGCUCAAAA	521
AS521-M1			UAUGGACCU
S471-	AUAUUUUGAGCAUAAUAUGAAGACT	471	AGUCUUCAUAUUAUGCUC	522
AS522-M1			AAAAUAUGG
S52-AS244-	GCAGCAAAUAUUGAUUUCUACAACC	52	GGUUGUAGAAAUCAAUAU	244
M1			UUGCUGCAC
S55-AS247-	GCAAAUAUUGAUUUCUACAACCATC	55	GAUGGUUGUAGAAAUCAA	247
M1			UAUUUGCUG
S75-AS267-	GGUUUCUGAAAUAACAGCAAUAGAA	75	UUCUAUUGCUGUUAUUUC	267
M1			AGAAACCGU
S77-AS269-	CUGAAAUAACAGCAAUAGAAGCUGA	77	UCAGCUUCUAUUGCUGUU	269
M1			AUUUCAGAA
S472-	ACGGCUUGAAUGUUAAGAAAUUUTC	472	GAAAAUUUCUUAACAUUC	523
AS523-M1			AAGCCGUUU
S473-	CGGCUUGAAUGUUAAGAAAUUUUCA	473	UGAAAAUUUCUUAACAUU	524
AS524-M1			CAAGCCGUU
S80-AS272-	GGCUUGAAUGUUAAGAAAUUUUCAG	80	CUGAAAAUUUCUUAACAU	272
M1			UCAAGCCGU
S81-AS273-	GCUUGAAUGUUAAGAAAUUUUCAGC	81	GCUGAAAAUUUCUUAACA	273
M1			UUCAAGCCG
S82-AS274-	CUUGAAUGUUAAGAAAUUUUCAGCA	82	UGCUGAAAAUUUCUUAAC	274
M1			AUUCAAGCC
S83-AS275-	UUGAAUGUUAAGAAAUUUUCAGCAG	83	CUGCUGAAAAUUUCUUAA	275
M1			CAUUCAAGC
S84-AS276-	UGAAUGUUAAGAAAUUUUCAGCAGT	84	ACUGCUGAAAAUUUCUUA	276
M1			ACAUUCAAG
S85-AS277-	GAAUGUUAAGAAAUUUUCAGCAGTG	85	CACUGCUGAAAAUUUCUU	277
M1			AACAUUCAA
S474-	AAUGUUAAGAAAUUUUCAGCAGUGC	474	GCACUGCUGAAAAUUUCU	525
AS525-M1			UAACAUUCA
S86-AS278-	AUGUUAAGAAAUUUUCAGCAGUGCA	86	UGCACUGCUGAAAAUUUC	278
M1			UUAACAUUC
S475-	UGUUAAGAAAUUUUCAGCAGUGCAT	475	AUGCACUGCUGAAAAUUU	526
AS526-M1			CUUAACAUU
S476-	GUUAAGAAAUUUUCAGCAGUGCATG	476	CAUGCACUGCUGAAAAUU	527
AS527-M1			UCUUAACAU
S143-	GGAGUAUUUCCAUCAUACUAUGAAC	143	GUUCAUAGUAUGAUGGAA	335
AS335-M1			AUACUCCAA
S181-	GCUGCAUGGUGAAUAUAAGAACUGA	181	UCAGUUCUUAUAUUCACC	373
AS373-M1			AUGCAGCUU
S182-	CUGCAUGGUGAAUAUAAGAACUGAA	182	UUCAGUUCUUAUAUUCAC	374
AS374-M1			CAUGCAGCU
S477-	UGCAUGGUGAAUAUAAGAACUGAAT	477	AUUCAGUUCUUAUAUUCA	528
AS528-M1			CCAUGCAGC
S183-	GCAUGGUGAAUAUAAGAACUGAATT	183	AAUUCAGUUCUUAUAUUC	375
AS375-M1			ACCAUGCAG
S184-	CAUGGUGAAUAUAAGAACUGAAUTC	184	GAAUUCAGUUCUUAUAUU	376
AS376-M1			CACCAUGCA
S185-	AUGGUGAAUAUAAGAACUGAAUUCT	185	AGAAUUCAGUUCUUAUAU	377
AS377-M1			UCACCAUGC
S186-	UGGUGAAUAUAAGAACUGAAUUCTA	186	UAGAAUUCAGUUCUUAUA	378
AS378-M1			UUCACCAUG
S187-	GGUGAAUAUAAGAACUGAAUUCUAC	187	GUAGAAUUCAGUUCUUAU	379
AS379-M1			AUUCACCAU
S188-	GUGAAUAUAAGAACUGAAUUCUACA	188	UGUAGAAUUCAGUUCUUA	380
AS380-M1			UAUUCACCA
S478-	UGAAUAUAAGAACUGAAUUCUACAT	478	AUGUAGAAUUCAGUUCUU	529
AS529-M1			AUAUUCACC
S479-	GAAUAUAAGAACUGAAUUCUACATG	479	CAUGUAGAAUUCAGUUCU	530
AS530-M1			UAUAUUCAC
S189-	AAUAUAAGAACUGAAUUCUACAUGT	189	ACAUGUAGAAUUCAGUUC	381
AS381-M1			UUAUAUUCA
S480-	CAAAGUAAGACUAAUUAUUUAAAAT	480	AUUUUAAAUAAUUAGUCU	531
AS531-M1			UACUUUGCU
S481-	AAAGUAAGACUAAUUAUUUAAAATA	481	UAUUUUAAAUAAUUAGUC	532
AS532-M1			UUACUUUGC
S482-	AGAAAUUGAGUGAAUGACAAUUUTG	482	CAAAAUUGUCAUUCACUCA	533
AS533-M1			AUUUCUUC
S483-	AAAUUGAGUGAAUGACAAUUUUGTA	483	UACAAAAUUGUCAUUCACU	534
AS534-M1			CAAUUUCU
S484-	AUUGAGUGAAUGACAAUUUUGUAAT	484	AUUACAAAAUUGUCAUUC	535
AS535-M1			ACUCAAUUU
S485-	AAUGACAAUUUUGUAAUUUAGGATA	485	UAUCCUAAAUUACAAAAUU	536
AS536-M1			GUCAUUCA
S486-	AAGUGUUUUUAAAAUGGUGAAUUTA	486	UAAAUUCACCAUUUUAAAA	537
AS537-M1			ACACUUUU
S487-	AGUGUUUUUAAAAUGGUGAAUUUAA	487	UUAAAUUCACCAUUUUAA	538
AS538-M1			AAACACUUU
S488-	CUUACUCUGUUUAUUUUUAAAUGAT	488	AUCAUUUAAAAAUAAACAG	539
AS539-M1			AGUAAGAG
S489-	CUCUGUUUAUUUUUAAAUGAUCATC	489	GAUGAUCAUUUAAAAAUA	540
AS540-M1			AACAGAGUA
S490-	UCUGUUUAUUUUUAAAUGAUCAUCA	490	UGAUGAUCAUUUAAAAAU	541
AS541-M1			AAACAGAGU
S491-	GUUUAUUUUUAAAUGAUCAUCAUAA	491	UUAUGAUGAUCAUUUAAA	542
AS542-M1			AAUAAACAG
S492-	AUCAUCAUAAUCCUUUGCUUACUAT	492	AUAGUAAGCAAAGGAUUA	543
AS543-M1			UGAUGAUCA
S493-	GUGCACUACCUACAUUUUUUAAATA	493	UAUUUAAAAAAUGUAGGU	544
AS544-M1			AGUGCACAU
S494-	GCUAGGUUUUUACUGAUUAUUUUCA	494	UGAAAAUAAUCAGUAAAAA	545
AS545-M1			CCUAGCUA
S495-	CUAGGUUUUUACUGAUUAUUUUCAT	495	AUGAAAAUAAUCAGUAAAA	546
AS546-M1			ACCUAGCU
S496-	AGGUUUUUACUGAUUAUUUUCAUTT	496	AAAUGAAAAUAAUCAGUAA	547
AS547-M1			AAACCUAG
S497-	CUGAUUAUUUUCAUUUUUCACAUGC	497	GCAUGUGAAAAAUGAAAA	548
AS548-M1			UAAUCAGUA
S498-	AUGGACAUUUAUGUCACUUUUGAAA	498	UUUCAAAAGUGACAUAAA	549
AS549-M1			UGUCCAUUA
S499-	GACAUUUAUGUCACUUUUGAAAUCT	499	AGAUUUCAAAAGUGACAU	550
AS550-M1			AAAUGUCCA
S500-	ACAUUUAUGUCACUUUUGAAAUCTA	500	UAGAUUUCAAAAGUGACA	551
AS551-M1			UAAAUGUCC
S501-	UAGAAUUGAUGUUGUAAUUAAUGCA	501	UGCAUUAAUUACAACAUCA	552
AS552-M1			AUUCUAGA
S502-	AGAAUUGAUGUUGUAAUUAAUGCAA	502	UUGCAUUAAUUACAACAUC	553
AS553-M1			AAUUCUAG
S503-	GAAUUGAUGUUGUAAUUAAUGCAAG	503	CUUGCAUUAAUUACAACAU	554
AS554-M1			CAAUUCUA
S504-	ACCAUCUUACUGUAACAUUUUUCTA	504	UAGAAAAAUGUUACAGUA	555
AS555-M1			AGAUGGUGG
S505-	CAUCUUACUGUAACAUUUUUCUATT	505	AAUAGAAAAAUGUUACAG	556
AS556-M1			UAAGAUGGU
S506-	UCUUACUGUAACAUUUUUCUAUUGT	506	ACAAUAGAAAAAUGUUACA	557
AS557-M1			GUAAGAUG
S507-	CUUACUGUAACAUUUUUCUAUUGTT	507	AACAAUAGAAAAAUGUUAC	558
AS558-M1			AGUAAGAU
5508-	UUACUGUAACAUUUUUCUAUUGUTT	508	AAACAAUAGAAAAAUGUUA	559
AS559-M1			CAGUAAGA
S509-	ACUGUAACAUUUUUCUAUUGUUUAA	509	UUAAACAAUAGAAAAAUG	560
AS560-M1			UUACAGUAA
S510-	CUGUAACAUUUUUCUAUUGUUUAAA	510	UUUAAACAAUAGAAAAAU	561
AS561-M1			GUUACAGUA
S511-	UGUAACAUUUUUCUAUUGUUUAAAT	511	AUUUAAACAAUAGAAAAA	562
AS562-M1			UGUUACAGU
S512-	GUAACAUUUUUCUAUUGUUUAAATA	512	UAUUUAAACAAUAGAAAA	563
AS563-M1			AUGUUACAG
S513-	UAACAUUUUUCUAUUGUUUAAAUAG	513	CUAUUUAAACAAUAGAAAA	564
AS564-M1			AUGUUACA
S514-	AACAUUUUUCUAUUGUUUAAAUAGA	514	UCUAUUUAAACAAUAGAA	565
AS565-M1			AAAUGUUAC
S515-	ACAUUUUUCUAUUGUUUAAAUAGAA	515	UUCUAUUUAAACAAUAGA	566
AS566-M1			AAAAUGUUA
S516-	CAUUUUUCUAUUGUUUAAAUAGAAA	516	UUUCUAUUUAAACAAUAG	567
AS567-M1			AAAAAUGUU
S517-	GUCAAUCUUCAUAGAUGAUAACUTG	517	CAAGUUAUCAUCUAUGAA	568
AS568-M1			GAUUGACCA
S569-	GGCAUCUAUACUGUGAUUCAGCAGCC	569	UGAAUCACAGUAUAGAUG	575
AS575-M14	GAAAGGCUGC		CCGG
S569-	GGCAUCUAUACUGUGAUUCAGCAGCC	569	UGAAUCACAGUAUAGAUG	575
AS575-M15	GAAAGGCUGC		CCGG
S570-	GCAUCUAUACUGUGAUUCAGGCAGCC	570	CUGAAUCACAGUAUAGAU	576
AS576-M14	GAAAGGCUGC		GCGG
S570-	GCAUCUAUACUGUGAUUCAGGCAGCC	570	CUGAAUCACAGUAUAGAU	576
AS576-M15	GAAAGGCUGC		GCGG
S571-	GCAGCAAAUAUUGAUUUCUAGCAGCC	571	UAGAAAUCAAUAUUUGCU	577
AS577-M14	GAAAGGCUGC		GCGG
S571-	GCAGCAAAUAUUGAUUUCUAGCAGCC	571	UAGAAAUCAAUAUUUGCU	577
AS577-M15	GAAAGGCUGC		GCGG
S572-	CUGAAAUAACAGCAAUAGAAGCAGCC	572	UUCUAUUGCUGUUAUUUC	578
AS578-M15	GAAAGGCUGC		AGGG
S573-	ACGGCUUGAAUGUUAAGAAAGCAGCC	573	UUUCUUAACAUUCAAGCC	579
AS579-M15	GAAAGGCUGC		GUGG
S574-	AGUGUUUUUAAAAUGGUGAAGCAGC	574	UUCACCAUUUUAAAAACAC	580
AS580-M14	CGAAAGGCUGC		UGG
S574-	AGUGUUUUUAAAAUGGUGAAGCAGC	574	UUCACCAUUUUAAAAACAC	580
AS580-M15	CGAAAGGCUGC		UGG
S385-	AGGUGCAUUUUGGAAGAUGGGCAGC	385	CCAUCUUCCAAAAUGCACC	417
AS417-M16	CGAAAGGCUGC		UGG
S389-	AAGGAAGUCCUUAUGUGGUAGCAGCC	389	UACCACAUAAGGACUUCCU	421
AS421-M17	GAAAGGCUGC		UCU
S391-	AUGAUAUGCUGAUAUUUGGAGCAGC	391	UCCAAAUAUCAGCAUAUCA	425
AS425-M16	CGAAAGGCUGC		UUG
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAAUAUUUG	429
AS429-M16	GAAAGGCUGC		CUGC
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	436
AS436-M16	GAAAGGCUGC		GCC
S397-	CUUGAAUGUUAAGAAAUUUAGCAGCC	397	UAAAUUUCUUAACAUUCA	586
AS586-M18	GAAAGGCUGC		AGGG
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAUUUCUUAACAUUCAA	587
AS587-M19	GAAAGGCUGC		GGG
S395-	CCACGGUUUCUGAAAUAACAGCAGCC	395	UGUUAUUUCAGAAACCGU	433
AS433-M17	GAAAGGCUGC		GGUG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	438
AS438-M16	GAAAGGCUGC		CAAG
S401-	CGAGGUCAUUUCUAUGGUCAGCAGCC	401	UGACCAUAGAAAUGACCUC	442
AS442-M17	GAAAGGCUGC		GAA
S405-	CUUGGAGUAUUUCCAUCAUAGCAGCC	405	UAUGAUGGAAAUACUCCA	448
AS448-M17	GAAAGGCUGC		AGAU
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	461
AS461-M17	GAAAGGCUGC		CUUC
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	588
AS588-M20	GAAAGGCUGC		CUGG
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UCACCAUGCAGCUUUUUC	589
AS589-M21	GAAAGGCUGC		UGG
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	461
AS461-M22	GAAAGGCUGC		CUUC
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	465
AS465-M16	GAAAGGCUGC		CAUG
S393-	AGCAAAUAUUGAUUUCUACAGCAGCC	393	UGUAGAAAUCAAUAUUUG	589
AS589-M18	GAAAGGCUGC		CUGG
S397-	CUUGAAUGUUAAGAAAUUUAGCAGCC	397	UAAAUUUCUUAACAUUCA	586
AS586-M23	GAAAGGCUGC		AGGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M18	GAAAGGCUGC		CAGG
S413-	AGAAAAAGCUGCAUGGUGAAGCAGCC	413	UUCACCAUGCAGCUUUUU	588
AS588-M20	GAAAGGCUGC		CUGG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	591
AS591-M18	GAAAGGCUGC		CAGG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	591
AS591-M23	GAAAGGCUGC		CAGG
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	592
AS592-M24	GAAAGGCUGC		GGG
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	592
AS592-M25	GAAAGGCUGC		GGG
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	592
AS592-M26	GAAAGGCUGC		GGG
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	592
AS592-M27	GAAAGGCUGC		GGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M24	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M25	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M26	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M27	GAAAGGCUGC		CAGG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	591
AS591-M28	GAAAGGCUGC		CAGG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	591
AS591-M29	GAAAGGCUGC		CAGG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	591
AS591-M30	GAAAGGCUGC		CAGG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	591
AS591-M31	GAAAGGCUGC		CAGG
S397-	CUUGAAUGUUAAGAAAUUUUGCAGCC	397	AAAAUUUCUUAACAUUCAA	592
AS592-M32	GAAAGGCUGC		GGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M32	GAAAGGCUGC		CAGG
S415-	UGGUGAAUAUAAGAACUGAAGCAGCC	415	UUCAGUUCUUAUAUUCAC	591
AS591-M33	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M34	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M35	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M28	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M36	GAAAGGCUGC		CAGG
S581-	GAAUGUUAAGAAAUUUUCAGCAGCCG	581	UGAAAAUUUCUUAACAUU	593
AS593-M37	AAAGGCUGC		CGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M38	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M39	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M40	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M41	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M42	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M43	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M24	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M44	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M28	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M45	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M40	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M46	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M38	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M46	GAAAGGCUGC		CAGG
S571-	GCAGCAAAUAUUGAUUUCUAGCAGCC	571	UAGAAAUCAAUAUUUGCU	577
AS577-M47	GAAAGGCUGC		GCGG
S573-	ACGGCUUGAAUGUUAAGAAAGCAGCC	573	UUUCUUAACAUUCAAGCC	579
AS579-M47	GAAAGGCUGC		GUGG
S582-	UGCAUGAGUUUCAAAAUCUAGCAGCC	582	UAGAUUUUGAAACUCAUG	594
AS594-M47	GAAAGGCUGC		CAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M47	GAAAGGCUGC		GAGG
S584-	AGACUAAUUAUUUAAAAUAAGCAGCC	584	UUAUUUUAAAUAAUUAGU	596
AS596-M47	GAAAGGCUGC		CUGG
S574-	AGUGUUUUUAAAAUGGUGAAGCAGC	574	UUCACCAUUUUAAAAACAC	580
AS580-M47	CGAAAGGCUGC		UGG
S585-	AGAAUUGAUGUUGUAAUUAAGCAGCC	585	UUAAUUACAACAUCAAUUC	597
AS597-M47	GAAAGGCUGC		UGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M48	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M49	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M50	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M51	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M52	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M53	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M54	GAAAGGCUGC		CAGG
S399-	UGAAUGUUAAGAAAUUUUCAGCAGCC	399	UGAAAAUUUCUUAACAUU	590
AS590-M55	GAAAGGCUGC		CAGG
S573-	ACGGCUUGAAUGUUAAGAAAGCAGCC	573	UUUCUUAACAUUCAAGCC	579
AS579-M56	GAAAGGCUGC		GUGG
S573-	ACGGCUUGAAUGUUAAGAAAGCAGCC	573	UUUCUUAACAUUCAAGCC	579
AS579-M57	GAAAGGCUGC		GUGG
S573-	ACGGCUUGAAUGUUAAGAAAGCAGCC	573	UUUCUUAACAUUCAAGCC	579
AS579-M50	GAAAGGCUGC		GUGG
S573-	ACGGCUUGAAUGUUAAGAAAGCAGCC	573	UUUCUUAACAUUCAAGCC	579
AS579-M51	GAAAGGCUGC		GUGG
S573-	ACGGCUUGAAUGUUAAGAAAGCAGCC	573	UUUCUUAACAUUCAAGCC	579
AS579-M52	GAAAGGCUGC		GUGG
S573-	ACGGCUUGAAUGUUAAGAAAGCAGCC	573	UUUCUUAACAUUCAAGCC	579
AS579-M53	GAAAGGCUGC		GUGG
S573-	ACGGCUUGAAUGUUAAGAAAGCAGCC	573	UUUCUUAACAUUCAAGCC	579
AS579-M54	GAAAGGCUGC		GUGG
S573-	ACGGCUUGAAUGUUAAGAAAGCAGCC	573	UUUCUUAACAUUCAAGCC	579
AS579-M55	GAAAGGCUGC		GUGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M56	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M58	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M50	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M51	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M52	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M53	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M54	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M55	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M59	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M60	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M61	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M62	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M63	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M64	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M65	GAAAGGCUGC		GAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M66	GAAAGGCUGC		GAGG
S574-	AGUGUUUUUAAAAUGGUGAAGCAGC	574	UUCACCAUUUUAAAAACAC	580
AS580-M56	CGAAAGGCUGC		UGG
S574-	AGUGUUUUUAAAAUGGUGAAGCAGC	574	UUCACCAUUUUAAAAACAC	580
AS580-M58	CGAAAGGCUGC		UGG
S574-	AGUGUUUUUAAAAUGGUGAAGCAGC	574	UUCACCAUUUUAAAAACAC	580
AS580-M50	CGAAAGGCUGC		UGG
S574-	AGUGUUUUUAAAAUGGUGAAGCAGC	574	UUCACCAUUUUAAAAACAC	580
AS580-M51	CGAAAGGCUGC		UGG
S574-	AGUGUUUUUAAAAUGGUGAAGCAGC	574	UUCACCAUUUUAAAAACAC	580
AS580-M52	CGAAAGGCUGC		UGG
S574-	AGUGUUUUUAAAAUGGUGAAGCAGC	574	UUCACCAUUUUAAAAACAC	580
AS580-M53	CGAAAGGCUGC		UGG
S574-	AGUGUUUUUAAAAUGGUGAAGCAGC	574	UUCACCAUUUUAAAAACAC	580
AS580-M54	CGAAAGGCUGC		UGG
S574-	AGUGUUUUUAAAAUGGUGAAGCAGC	574	UUCACCAUUUUAAAAACAC	580
AS580-M55	CGAAAGGCUGC		UGG
S612-	UGGGAGGUAUGAGUUUUCAAGCAGC	612	UUGAAAACUCAUACCUCCC	620
AS620-M5	CGAAAGGCUGC		AGG
S613-	AGGAAAAGUUUGGAAAAAAAGCAGCC	613	UUUUUUUCCAAACUUUUC	621
AS621-M5	GAAAGGCUGC		CUGG
S614-	UGGUCAAUCUUCAUAGAUGAGCAGCC	614	UCAUCUAUGAAGAUUGAC	622
AS622-M5	GAAAGGCUGC		CAGG
S615-	GGUUUCUGAAAUAACAGCAAGCAGCC	615	UUGCUGUUAUUUCAGAAA	623
AS623-M5	GAAAGGCUGC		CCGG
S572-	CUGAAAUAACAGCAAUAGAAGCAGCC	572	UUCUAUUGCUGUUAUUUC	578
AS578-M5	GAAAGGCUGC		AGGG
S616-	GUGAAUAUAAGAACUGAAUUGCAGCC	616	AAUUCAGUUCUUAUAUUC	624
AS624-M5	GAAAGGCUGC		ACGG
S617-	AUUGAGUGAAUGACAAUUUUGCAGCC	617	AAAAUUGUCAUUCACUCAA	625
AS625-M5	GAAAGGCUGC		UGG
S618-	AAUGACAAUUUUGUAAUUUAGCAGCC	618	UAAAUUACAAAAUUGUCA	626
AS626-M5	GAAAGGCUGC		UUGG
S585-	AGAAUUGAUGUUGUAAUUAAGCAGCC	585	UUAAUUACAACAUCAAUUC	597
AS597-M5	GAAAGGCUGC		UGG
S619-	GAAUUGAUGUUGUAAUUAAUGCAGC	619	AUUAAUUACAACAUCAAUU	627
AS627-M5	CGAAAGGCUGC		CGG
S573-	ACGGCUUGAAUGUUAAGAAAGCAGCC	573	UUUCUUAACAUUCAAGCC	579
AS579-M5	GAAAGGCUGC		GUGG
S582-	UGCAUGAGUUUCAAAAUCUAGCAGCC	582	UAGAUUUUGAAACUCAUG	594
AS594-M5	GAAAGGCUGC		CAGG
S583-	UCGAGAUGAUCUAACAAUUAGCAGCC	583	UAAUUGUUAGAUCAUCUC	595
AS595-M5	GAAAGGCUGC		GAGG
S584-	AGACUAAUUAUUUAAAAUAAGCAGCC	584	UUAUUUUAAAUAAUUAGU	596
AS596-M5	GAAAGGCUGC		CUGG

The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

It should be appreciated that, in some embodiments, sequences presented in the sequence listing may be referred to in describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and/or one or more modified nucleotides and/or one or more modified internucleotide linkages and/or one or more other modification compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. An oligonucleotide for reducing expression of GYS2, the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and a sense strand of 36 to 40 nucleotides in length, wherein the sense strand forms a duplex region with the antisense strand, and wherein:

the antisense strand has a region of complementarity to a target sequence of GYS2 as set forth in any one of SEQ ID NOs: 599-608, wherein the region of complementarity is at least 15 contiguous nucleotides in length; and

the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 385-416, 569-574, 581-585, and 612-619.

2. The oligonucleotide of claim 1, wherein the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 417-466, 575-580, 586-598, and 620-627.

3. The oligonucleotide of claim 1, wherein the sense strand comprises at its 3′-end a stem-loop set forth as: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2 of 3 to 5 nucleotides in length.

4. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a 3′-overhang sequence of one or more nucleotides in length, wherein the 3′-overhang sequence is present on the antisense strand, the sense strand, or the antisense strand and sense strand.

5. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one modified nucleotide and wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid.

6. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one modified internucleotide linkage, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

7. The oligonucleotide of claim 1, wherein the duplex region formed by the sense strand and the antisense strand is at least 19 nucleotides in length.

8. The oligonucleotide of claim 1, wherein the region of complementarity of the antisense strand to the target sequence of GYS2 is at least 19 contiguous nucleotides in length.

9. The oligonucleotide of claim 1, wherein the antisense strand is 21 to 27 nucleotides in length.

10. The oligonucleotide of claim 1, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.

11. The oligonucleotide of claim 10, wherein the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

12. The oligonucleotide of claim 1, wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

13. A composition comprising the oligonucleotide of claim 1 and an excipient.

14. An oligonucleotide for reducing expression of GYS2, the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length, wherein the sense strand forms a duplex region with the antisense strand, and wherein:

the antisense strand has a region of complementarity to a target sequence of GYS2 as set forth in any one of SEQ ID NOs: 599-608, wherein the region of complementarity is at least 15 contiguous nucleotides in length; and

the sense strand comprises at its 3′-end a stem-loop set forth as: S1-L-S2, wherein S1 is complementary to S2; L forms a loop between S1 and S2; and L is a tetraloop.

15. The oligonucleotide of claim 14, wherein the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 417-466, 575-580, 586-598, and 620-627.

16. The oligonucleotide of claim 14, wherein the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 385-416, 569-574, 581-585, and 612-619.

17. The oligonucleotide of claim 14, wherein the duplex region formed by the sense strand and the antisense strand is at least 19 nucleotides in length.

18. The oligonucleotide of claim 14, wherein the region of complementarity of the antisense strand to the target sequence of GYS2 is at least 19 contiguous nucleotides in length.

19. The oligonucleotide of claim 14, wherein the antisense strand is 21 to 27 nucleotides in length.

20. The oligonucleotide of claim 14, wherein the oligonucleotide comprises a 3′-overhang sequence of one or more nucleotides in length, wherein the 3′-overhang sequence is present on the antisense strand, the sense strand, or the antisense strand and sense strand.

21. The oligonucleotide of claim 14, wherein the oligonucleotide comprises at least one modified nucleotide and wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid.

22. The oligonucleotide of claim 14, wherein the oligonucleotide comprises at least one modified internucleotide linkage, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

23. The oligonucleotide of claim 14, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.

24. The oligonucleotide of claim 23, wherein the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

25. The oligonucleotide of claim 14, wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

26. A composition comprising the oligonucleotide of claim 14 and an excipient.

27. An oligonucleotide for reducing expression of GYS2, wherein:

the antisense strand has a region of complementarity to a target sequence of GYS2 as set forth in any one of SEQ ID NOs: 599-608, wherein the region of complementarity is at least 15 contiguous nucleotides in length;

the oligonucleotide comprises a 3′-overhang sequence of two nucleotides in length, wherein the 3′-overhang sequence is present on the antisense strand;

the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and antisense strand form a duplex of 21 nucleotides in length;

the oligonucleotide comprises at least one modified nucleotide and wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid; and

wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

28. The oligonucleotide of claim 27, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.

29. The oligonucleotide of claim 28, wherein the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

30. The oligonucleotide of claim 27, wherein the duplex region formed by the sense strand and the antisense strand is at least 19 nucleotides in length.

31. The oligonucleotide of claim 27, wherein the region of complementarity of the antisense strand to the target sequence of GYS2 is at least 19 contiguous nucleotides in length.

32. The oligonucleotide of claim 27, wherein the oligonucleotide comprises at least one modified internucleotide linkage, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

33. The oligonucleotide of claim 27, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.

34. The oligonucleotide of claim 33, wherein the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

35. A composition comprising the oligonucleotide of claim 27 and an excipient.